Author Response

The following is the authors’ response to the original reviews.

We would like to extend our gratitude to the reviewers for their meticulous analysis and constructive feedback on our manuscript. We have revised our paper based on the suggestions regarding supporting literature and the theory behind CAPs along with detailed insights regarding our methods. Their suggestions have been extremely useful in strengthening the clarity and rigor of our manuscript.

Reviewer #1 (Recommendations For The Authors):

(1) There are no obvious problems with this paper and it is relatively straightforward. There are some challenges that I would like to suggest. These variants have multiple mutations, so it would be interesting if you could drill down to find out which mutation is the most important for the collective changes reported here. I would like to see a sequence alignment of these variants, perhaps in the supplemental material, just to get some indication of the extent of mutations involved.

Finding the most important mutation within a set is a tricky question, as each mutation changes the way future mutations will affect function due to epistasis. Indeed, this is what we aim to explore in this work. To illustrate this point, we included a new supplementary figure S5A. Three critical mutations that emerged quickly, and were frequently observed in other dominant variants, were S477N, T478K, and N501Y. Thus, we computed the EpiScore values of these three mutations, with several critical residues contributing to hACE2 binding. The EpiScore distribution indicates that residues 477, 478, and 501 have strong epistatic (i.e., non-additive) interactions, as indicated by EpiScore values above 2.0.

To further investigate these epistatic interactions, we first conducted MD simulations and computed the DFI profile of these three single mutants. We analyzed how different the DFI scores of the hACE2 binding interface residues of the RBD are, across three single mutants with Omicron, Delta, and Omicron XBB variants (Fig S5B). Fig S5B shows how mutations at these particular sites affect the binding interface DFI in various backgrounds, as the three mutations are also observed in the Omicron, XBB, and XBB 1.5 variants. If the difference in the DFI profile of the mutant and the given variant is close to 0, then we could safely state that this mutation affected the variant the most. However, what we observe is quite the opposite: the DFI profile of the mutation is significantly different in different variant backgrounds. While these mutations may change overall behavior, their individual contributions to overall function are more difficult to pin down because overall function is dependent on the non-additive interactions between many different residues.

Author response image 1.

(A) Three critical mutations that emerged quickly, and were frequently observed in other dominant variants, were S477N, T478K, and N501Y. EpiScores of sites 477, 478, and 501 with one another are shown with k = the binding interface of the open chain. These residues are highly epistatic, producing higher responses than expected when perturbed together. (B) The difference in the dynamic flexibility profiles between the single mutants and the most common variants for the hACE2 binding residues of the RBD. DFI profiles exhibit significant variation from zero, and also show different flexibility in each background variant, highlighting the critical non-additive interactions of the other mutation in the given background variant. Thus, these three critical mutations, impacting binding affinity, do not solely contribute to the binding. There are epistatic interactions with the other mutations in VOCs that shape the dynamics of the binding interface to modulate binding affinity with hACE2.

As we discussed above, while the epistatic interactions are crucial and the collective impact of the mutations shape the mutational landscape of the spike protein, we would like note that mutation S486P is one of the critical mutations we identify, modulating both antibody and hACE2 binding and our analysis reveals the strong non-additive interactions with the other mutational sites. This mutational site appears in both XBB1.5 and earlier Omicron strains which highlights its importance in functional evolution of the spike protein. CAPs 346R, 486F, and 498Q also may be important, as they have a high EpiScore, indicating critical epistatic interaction with many mutation sites.

Regarding to the suggestion about presenting the alignment of the different variants, we have attached a mutation table, highlighting the mutated residues for each strain compared to the reference sequence as supplemental Figure S1 along with the full alignment file.

(2) Also, I am wondering if it would be possible to insert some of these flexibilities and their correlations directly into the elastic network models to enable a simpler interpretation of these results. I realize this is beyond the scope of the present work, but such an effort might help in understanding these relatively complex effects.

This is great suggestion. A similar analysis has been performed for different proteins by Mcleash (See doi: 10.1016/j.bpj.2015.08.009) by modulating the spring constants of specific position to alter specific flexibility and evaluate change in elastic free energy to identify critical mutation (in particular, allosteric mutation) sites. We will be happy to pursue this as future work.

Minor

(3) 1 typo on line 443 - should be binding instead of biding.

Fixed, thanks for spotting that.

(4) The two shades of blue in Fig. 4B were not distinguishable in my version.

To fix this, we have changed the overlapping residues between Delta and Omicron to a higher contrast shade of blue.

(5) Compensatory is often used in an entirely different way - additional mutations that help to recover native function in the presence of a deleterious mutation.

Although our previous study (Ose et al. 2022, Biophysical Journal) shows that compensatory mutations were generally additive, the two ideas are not one and the same. We thank the reviewer for pointing this out. Therefore, to clarify, we have now described our results in terms of dynamic additivity, rather than compensation.

Reviewer #2 (Recommendations For The Authors):

(1) The authors note that the identified CAPs overlap with those of others (Cagliani et al. 2020; Singh and Yi 2021; Starr, Zepeda, et al. 2022). In itself, this merits a deeper discussion and explicit indication of which positions are not identified. However, there is one point that I believe may represent a fundamental flaw in this study in that the calculation of EP from the alignment of S proteins ignores entirely the differences in the interacting interface with which S for different coronaviruses in the alignment interact in the different receptors in each host species. This may be the reason why so many "CAPs" are in the RBD. The authors should at the very least make a convincing case of why they are not simply detecting constraints imposed by the different interacting partners, at least in the case of positions within the RBD interface with ACE2. Another point that the authors should discuss is that ACE2 is not the only receptor that facilitates infection, TMPRSS2 and possibly others have been identified as well. The results should be discussed in light of this.

To begin with, we have now explicitly noted (on line 135) that “sites 478, 486, 498, and 681 have already been implicated in SARS-CoV-2 evolution, leaving the remaining 11 CAPs as undiscovered candidate sites for adaptation.” Evolutionary analyses are done using orthologous protein sequences, so there is no way to integrate information on different receptors in each host species in the calculation of EPs. However, we appreciate that the preponderance of CAPs in the RBD is likely due to different binding environments. We have added the following text (on line 83) to clarify our point: “Adaptation in this case means a virus which can successfully infect human hosts. As CAPs are unexpected polymorphisms under neutral theory, their existence implies a non-neutral effect. This can come in the form of functional changes (Liu et al. 2016) or compensation for functional changes (Ose et al. 2022). Therefore, we suspect that these CAPs, being unexpected changes from coronaviruses across other host species with different binding substrates, may be partially responsible for the functional change of allowing human infection.” This hypothesis is supported by the overlap of CAPs we identified with the positions identified in other studies (e.g., 478, 486, 498, and 681). Binding to TMPRSS2 and other substrates are also covered by this analysis as it is a measure of overall evolutionary fitness, rather than binding to any specific substrate. Our paper does focus on discussing hACE2 binding and mentions furin cleavage, but indeed lacks discussion on the role of TMPRSS2. We have added the following text to line 157: “Another host cell protease, TMPRSS2, facilitates viral attachment to the surface of target cells upon binding either to sites Arg815/Ser816, or Arg685/Ser686 which overlaps with the furin cleavage site 676-689, further emphasizing the importance of this area (Hoffmann et al. 2020b; Fraser et al. 2022).”

(2) Turning now to the computational methods utilized to study dynamics, I have serious reservations about the novelty of the results as well as the validity of the methodology. First of all, the authors mention the work of Teruel et al. (PLOS Comp Bio 2021) in an extremely superficial fashion and do not mention at all a second manuscript by Teruel et al. (Biorxiv 2021.12.14.472622 (2021)). However, the work by Teruel et al. identifies positions and specific mutations that affect the dynamics of S and the evolution of the SARS-CoV-2 virus in light of immune escape, ACE2 binding, and open and closed state dynamics. The specific differences in approach should be noted but the results specifically should be compared. This omission is evident throughout the manuscript. Several other groups have also published on the use of nomal-mode analysis methods to understand the Spike protein, among them Verkhivker et al., Zhou et al., Majumder et al., etc.

Thank you for your suggestions. Upon further examination of the listed papers, we have added citations to other groups employing similar methods. However, it's worth noting that the results of Teruel et al.'s studies are generally not directly comparable to our own. Particularly, they examine specific individual mutations and overall dynamical signatures associated with them, whereas our results are always considered in the context of epistasis and joint effects with CAPs, and all mutations belong to the common variants. Although important mutations may be highlighted in both cases, it is for very different reasons. Nevertheless, we provide a more detailed mention of the results of both studies. See lines 178, 255, and 393.

(3) The last concern that I have is with respect to the methodology. The dynamic couplings and the derived index (DCI) are entirely based on the use of the elastic network model presented which is strictly sequence-agnostic. Only C-alpha positions are taken into consideration and no information about the side-chain is considered in any manner. Of course, the specific sequence of a protein will affect the unique placement of C-alpha atoms (i.e., mutations affect structure), therefore even ANM or ENM can to some extent predict the effect of mutations in as much as these have an effect on the structure, either experimentally determined or correctly and even incorrectly modelled. However, such an approach needs to be discussed in far deeper detail when it comes to positions on the surface of a protein such that the reader can gauge if the observed effects are the result of modelling errors.

We would like to clarify that most of our results do not involve simulations of different variants, but rather how characteristic mutation sites for those variants contribute to overall dynamics. For the full spike, we operate on only two simulations: open and closed. When we do analyze different variants, starting on line 438, the observed difference does not come from the structure, but from the covariance matrix obtained from molecular dynamics (MD) simulations, which are sensitive to single amino acid changes.

Reviewer #3 (Recommendations For The Authors):

(1) On line 99 there is a misspelling, 'withing'.

It has been fixed. Thanks for spotting that.

(2) Some graphical suggestions to make the figures easier to read:

In Figure 1C, a labeled circle around the important sites, the receptor binding domain, and the Furin cleavage site, would help the reader orient themselves. Moreover, it would make clear which CAPs are NOT in the noteworthy sites described in the text.

Good idea. We have added transparent spheres and labels to show hACE2 binding sites and Furin cleavage sites.

In Figure 2C the colors are a bit low contrast; moreover, there are multiple text sizes on the same figure which should perhaps be avoided to ensure legibility.

We have made yellow brighter and standardized font sizes.

Figure 3 is a bit dry, perhaps indicating in which bins the 'interesting' sites could be informative.

Thank you for the suggestion, but the overall goal of Figure 3 is to illustrate that the mutational landscape is governed by the equilibrium dynamics in which flexible sites undergo more mutations during the evolution of the CoV2 spike protein. Therefore, adding additional positional information may complicate our message.

Figure 4, the previous suggestions about readability apply.

We ensured same sized text and higher contrast colors.

Figure 5B, the residue labels are too small.

We increased the font size of the residue labels.

In Figure 8 maybe adding Delta to let the reader orient themselves would be helpful to the discussion.

Unfortunately, there is no single work that has experimentally quantified binding affinities towards hACE2 for all the variants. When we conducted the same analysis for the Delta variant in Figure 8, the experimental values were obtained from a different source (doi: 10.1016/j.cell.2022.01.001) and the values were significantly different from the experimental work we used for Omicron (Yue et al. 2023). When we could adjust based on the difference in experimentally measured binding affinity values of the original Wuhan strain in these two separate studies, we observed a similar correlation, as seen below. However, we think this might not be a proper representation. Therefore, we chose to keep the original figure.

Author response image 2.

The %DFI calculations for variants Delta, Omicron, XBB, and XBB 1.5. (A) %DFI profile of the variants are plotted in the same panel. The grey shaded areas and dashed lines indicate the ACE2 binding regions, whereas the red dashed lines show the antibody binding residues. (B) The sum of %DFI values of RBD-hACE2 interface residues. The trend of total %DFI with the log of Kd values overlaps with the one seen with the experiments. (C) The RBD antibody binding residues are used to calculate the sum of %DFI. The ranking captured with the total %DFI agrees with the susceptibility fold reduction values from the experiments.

(3) Replicas of the MD simulations would make the conclusions stronger in my opinion.

We ran a 1µs long simulation and performed convergence analysis for the MD simulations using the prior work (Sawle L, Ghosh K. 2016.) More importantly, we also evaluated the statistical significance of computed DFI values as explained in detail below (Please see the answer to question 3 of Reviewer #3 (Public Review):)

Reviewer #3 (Public Review):

(1) A longer discussion of how the 19 orthologous coronavirus sequences were chosen would be helpful, as the rest of the paper hinges on this initial choice.

The following explanation has been added on line 114: EP scores of the amino acid variants of the S protein were obtained using a Maximum Likelihood phylogeny (Kumar et al. 2018) built from 19 orthologous coronavirus sequences. Sequences were selected by examining available non-human sequences with a sequence identity of 70% or above to the human SARS CoV-2’s S protein sequence. This cutoff allows for divergence over evolutionary history such that each amino acid position had ample time to experience purifying selection, whilst limiting ourselves to closely related coronaviruses. (Figure 1A).

(2) The 'reasonable similarity' with previously published data is not well defined, nor there was any comment about some of the residues analyzed (namely 417-484). We have revised this part of the manuscript and add to the revised version.

We removed the line about reasonable similarity as it was vague, added a line about residues 417-484, and revised the text accordingly, starting on line 354.

(3) There seem to be no replicas of the MD simulations, nor a discussion of the convergence of these simulations. A more detailed description of the equilibration and production schemes used in MD would be helpful. Moreover, there is no discussion of how the equilibration procedure is evaluated, in particular for non-experts this would be helpful in judging the reliability of the procedure.

We opted for a single, extended equilibrium simulation to comprehensively explore the longterm behavior of the system. Given the specific nature of our investigation and resource constraints, a well-converged, prolonged simulation was deemed a practical and scientifically valid approach, providing a thorough understanding of the system's dynamics. (doi: 10.33011/livecoms.1.1.5957, https://doi.org/10.1146/annurev-biophys-042910-155255 )

We updated our methods section starting on line 605 with extended information about the MD simulations and the converge criteria for the equilibrium simulations. We also added a section that explains our analysis to check statistical significance of obtained DFI values.

Author Response

The following is the authors’ response to the original reviews.

Response to reviewer’s comments

Reviewer #1 (Public Review):

In this study, the structural characteristics of plant AlaDC and SerDC were analyzed to understand the mechanism of functional differentiation, deepen the understanding of substrate specificity and catalytic activity evolution, and explore effective ways to improve the initial efficiency of theanine synthesis.

On the basis of previous solid work, the authors successfully obtained the X-ray crystal structures of the precursors of theanine synthesis-CsAlaDC and AtSerDC, which are key proteins related to ethylamine synthesis, and found a unique zinc finger structure on these two crystal structures that are not found in other Group II PLP-dependent amino acid decarboxylases. Through a series of experiments, it is pointed out that this characteristic zinc finger motif may be the key to the folding of CsAlaDC and AtSerDC proteins, and this discovery is novel and prospective in the study of theine synthesis.

In addition, the authors identified Phe106 of CsAlaDC and Tyr111 of AtSerDC as key sites of substrate specificity by comparing substrate binding regions and identified amino acids that inhibit catalytic activity through mutation screening based on protein structure. It was found that the catalytic activity of CsAlaDCL110F/P114A was 2.3 times higher than that of CsAlaDC. At the same time, CsAlaDC and AtSerDC substrate recognition key motifs were used to carry out evolutionary analysis of the protein sequences that are highly homologous to CsAlaDC in embryos, and 13 potential alanine decarboxylases were found, which laid a solid foundation for subsequent studies related to theanine synthesis.

In general, this study has a solid foundation, the whole research idea is clear, the experimental design is reasonable, and the experimental results provide strong evidence for the author's point of view. Through a large number of experiments, the key links in the theanine synthesis pathway are deeply studied, and an effective way to improve the initial efficiency of theanine synthesis is found, and the molecular mechanism of this way is expounded. The whole study has good novelty and prospectivity, and sheds light on a new direction for the efficient industrial synthesis of theanine

Response: Thank you very much for taking time to review this manuscript. We appreciate all your insightful comments and constructive suggestions.

Reviewer #1 (Recommendations For The Authors):

(1) If some test methods are not original, references or method basis should be indicated.

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have added references for the enzymatic activity experiments performed to measure the synthesis of theanine in the revised manuscript.

(2) The conclusion is a little lengthy, and the summary of the whole study is not well condensed.

Response: Thank you very much for your valuable suggestions. We have refined the conclusion in the revised manuscript, and it is as follows:

In conclusion, our structural and functional analyses have significantly advanced understanding of the substrate-specific activities of alanine and serine decarboxylases, typified by CsAlaDC and AtSerDC. Critical amino acid residues responsible for substrate selection were identified—Tyr111 in AtSerDC and Phe106 in CsAlaDC—highlighting pivotal roles in enzyme specificity. The engineered CsAlaDC mutant (L110F/P114A) not only displayed enhanced catalytic efficiency but also substantially improved L-theanine yield in a synthetic biosynthesis setup with PsGS or GMAS. Our research expanded the repertoire of potential alanine decarboxylases through the discovery of 13 homologous enzyme candidates across embryophytic species and uncovered a special motif present in serine protease-like proteins within Fabale, suggesting a potential divergence in substrate specificity and catalytic functions. These insights lay the groundwork for the development of industrial biocatalytic processes, promising to elevate the production of L-theanine and supporting innovation within the tea industry.

Reviewer #2 (Public Review)

Summary:

The manuscript focuses on the comparison of two PLP-dependent enzyme classes that perform amino acyl decarboxylations. The goal of the work is to understand the substrate specificity and factors that influence the catalytic rate in an enzyme linked to theanine production in tea plants.

Strengths:

The work includes x-ray crystal structures of modest resolution of the enzymes of interest. These structures provide the basis for the design of mutagenesis experiments to test hypotheses about substrate specificity and the factors that control catalytic rate. These ideas are tested via mutagenesis and activity assays, in some cases both in vitro and in plants.

Weaknesses:

The manuscript could be more clear in explaining the contents of the x-ray structures and how the complexes studied relate to the reactant and product complexes. The structure and mechanism section would also be strengthened by including a diagram of the reaction mechanism and including context about reactivity. As it stands, much of the structural results section consists of lists of amino acids interacting with certain ligands without any explanation of why these interactions are important or the role they play in catalysis. The experiments testing the function of a novel Zn(II)-binding domain also have serious flaws. I don't think anything can be said at this point about the function of the Zn(II) due to a lack of key controls and problems with experimental design.

Response: Thank you very much for your thoughtful comments and feedback on our manuscript. We are pleased to hear that the work's strengths, such as the X-ray crystal structures and the mutagenesis experiments tied to the catalytic rate and substrate specificity, align with the goals of our research.

We recognize the areas identified for improvement and appreciate the suggestions provided. We have emphasized how we use the structural information obtained to infer the roles of key amino acid residues in the reaction. Additionally, we have added a diagram of the reaction mechanism in the Supplementary figure to provide clearer context on reactivity and improve the overall understanding of the catalytic process. Regarding the structural results section, we have included a discussion that contextualizes the list of amino acids and their interactions with the ligands by explaining their significance and roles in catalysis. We acknowledge the weaknesses you've pointed out in the experiments concerning the novel Zn(II)-binding domain, but we would like to clarify that the focus of our study was not primarily on the zinc structure. While we agree that there may be limitations in the experimental design and controls for the zinc binding domain, we believe that these flaws do not significantly impact the overall findings of the study. The experiment served as a preliminary exploration of the potential functionality of the domain, and further studies are required to fully understand its role and mechanism.

Reviewer #2 (Recommendations For The Authors):

(1) In addition to the points raised in the public review, it would be ideal to provide some context for the enzymatic characterization. Why are the differences in kinetic parameters for AlaDC and SerDC significant?

Response: Thank you for your comments and suggestions. The Km values for CsAlaDC and SerDCs are comparable, suggesting similar substrate affinities. However, CsAlaDC exhibits a significantly lower Vmax compared to AtSerDC and CsSerDC. This discrepancy implies that CsAlaDC and SerDCs may differ in the rates at which they convert substrate to product when saturated with substrate. SerDCs may have a faster turnover rate, meaning they convert substrate to product and release the enzyme more quickly, resulting in a higher Vmax. Differences in the stability or correct folding of the enzymes under assay conditions can also affect their Vmax. If SerDCs are more stable, they might maintain their catalytic activity better at higher substrate concentrations, contributing to a higher Vmax. We have added these to the part of “Enzymatic properties of CsAlaDC, AtSerDC, and CsSerDC” in our revised manuscript.

(2) Why is Phe106/Tyr111 pair critical for substrate specificity? Does the amino acid contact the side chain? It might be helpful to a reader to formulate a hypothesis for this interaction.

Response: Thank you for the question and comments. We conducted a comparison between the active sites of CsAlaDC and AtSerDC and observed a distinct difference in only two amino acids: F106 in CsAlaDC and Y111 in AtSerDC. The remaining amino acids were found to be identical. Expanding on previous research concerning Group II PLP-dependent amino acid decarboxylases, it was postulated and subsequently confirmed that these specific amino acids play a crucial role in substrate recognition. However, since we lack the structure of the enzyme-substrate complex, we are unable to elucidate the precise interactions occurring between the substrate and the amino acids at this particular site based solely on structural information.

(3) Line 55 - Define EA again.

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have redefined “EA” as the abbreviation for ethylamine in the revised manuscript.

(4) Line 58 - The meaning of "determined by the quality formation of tea" is not clear.

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have modified it in the revised manuscript.

(5) Line 65 - Missing words between "despite they".

Response: Thank you very much for your careful reading of the manuscript. We have corrected it in the revised manuscript.

(6) Line 67 - Need a reference for the statement about lower activity?

Response: Thank you for the question and comments. We have provided the following reference to support this statement in the revised manuscript.

Reference: Bai, P. et al. (2021) Biochemical characterization of specific Alanine Decarboxylase (ADC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis). BMC Biotechnol. 21,17.

(7) Line 100-101 - The meaning of "its closer relationship was Dicots plants." is not clear.

Response: We have revised the sentence in the revised manuscript, as follows: “Phylogenetic analysis indicated that CsAlaDC is homologous with SerDCs in Dicots plants.”

(8) Line 139 - Missing a word between "as well as" and "of".

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

(9) Line 142 - The usage of comprised here is not correct. It would be more correct to say "The overall architecture of CsAlaDC and AtSerDC is homodimeric with the two subunits...".

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

(10) Line 148-149 - I didn't understand the statement about the "N-terminal structures" Are these structures obtained from protein samples that have a truncated N-terminus?

Response: Group II PLP-dependent amino acid decarboxylases are comprised of three distinct structural domains: the N-terminal domain, the large domain, and the C-terminal domain. Each of these domains possesses unique structural features. Similarly, CsAlaDC and AtSerDC can also be classified into three structural domains based on their specific characteristics. To achieve more stable proteins for further experiments, we conducted truncation on both of these proteins. The truncated section pertains to a subsection of the N-terminal domain and is truncated from the protein's N-terminus.

(11) Line 153 - Say "is composed of" instead of "composes of".

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

(12) Line 156 - I didn't understand the statement about the cofactor binding process. What is the cofactor observed? And how can we say anything about the binding process from a single static structure of the enzyme? It might be better to say that the cofactor binding site is located at the subunit junction - but the identity of the cofactor still needs to be defined first.

Response: Thank you for your comments and suggestions. The cofactor mentioned here is PLP. We aim to elucidate the binding state of PLP at the active site, excluding the binding process. The description has been revised in the revised manuscript.

(13) Lines 157-158 - I didn't understand the conclusion about the roles of each monomer. In the images in Figure 3 - both monomers appear to bind PLP but the substrate is not present - so it's not clear how conclusions can be drawn about differential substrate binding in the two subunits.

Response: Thank you very much for your careful reading and valuable suggestions. The main idea we want to convey is that this protein possesses two active sites. At each active site, the two monomers carry out distinct functions. Of course, our previous conclusion is inaccurate due to the non-existence of the substrate. So, we have made the necessary amendments in the revised manuscript.

(14) Line 161 - I would say loop instead of ring.

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

(15) Line 165 - Please provide some references for this statement. It would also be ideal to state the proximity of the Zn-binding motif to the active site or otherwise provide some information about the role of the motif based on its location.

Response: Thank you for your comments and suggestions. We have provided the following references to support this statement in the revised manuscript.

Author response image 1.

(A) Structure of histidine decarboxylase. (B) Structure of glutamate decarboxylase.

Reference:

30 Komori, H. et al. (2012) Structural study reveals that Ser-354 determines substrate specificity on human Histidine Decarboxylase. J Biol Chem. 287, 29175-83.

31 Huang, J. et al. (2018) Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis. Biochem Biophys Res Co. 503, 1703-1709

In CsAlaDC, the zinc is positioned at a distance of 29.6 Å from the active center, whereas in AtSerDC, the zinc is situated 29 Å away from the active center. Hence, we hypothesize that this structure does not impact the enzyme's catalytic activity but might be correlated with its stability.

(16) Lines 166-178 - This paragraph appears to be a list of all of the interactions between the protein, PLP, and the EA product. It would be ideal to provide some text to explain why these interactions are important and what we can learn from them.

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have been conducting additional analysis on the functional roles of amino acid residues involved in the interaction between the active site and PLP. This analysis focuses on aiding PLP binding, determining its orientation, and understanding enzyme catalytic mechanisms. These details are mentioned in the revised manuscript.

(17) Line 192 - Bond not bound.

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have made corrections in the revised manuscript.

(18) Lines 201-207 - It would be ideal to verify that the inclusion of 5 mM DTT affects Zn binding. It's not clear to me that this reagent would necessarily disrupt Zn binding. Under certain circumstances, it could instead promote Zn association. For example, if the Cys ligands are oxidized initially but then become reduced? I don't think the current experiment really provides any insight into the role of the Zn.

Response: Thank you for your valuable insights regarding the role of DTT and its potential effects on Zn binding in our experiments. The main function of DTT is to protect or restore the reduced state of proteins and other biological molecules, particularly by disrupting the crosslinking formed by thiol (-SH) groups and disulfide bonds to maintain the function and structure of proteins. Therefore, the reason for DTT's inhibition of enzyme activity is unknown, and we cannot provide a reasonable explanation for this phenomenon. As a result, we have removed the section discussing the inhibition of enzyme activity by DTT in our revised manuscript.

Reviewer #3 (Public Review):

In the manuscript titled "Structure and Evolution of Alanine/Serine Decarboxylases and the Engineering of Theanine Production," Wang et al. solved and compared the crystal structures of Alanine Decarboxylase (AlaDC) from Camellia sinensis and Serine Decarboxylase (SerDC) from Arabidopsis thaliana. Based on this structural information, the authors conducted both in vitro and in vivo functional studies to compare enzyme activities using site-directed mutagenesis and subsequent evolutionary analyses. This research has the potential to enhance our understanding of amino acid decarboxylase evolution and the biosynthetic pathway of the plant-specialized metabolite theanine, as well as to further its potential applications in the tea industry. Response: Thank you very much for taking the time to review this manuscript. We appreciate all your insightful comments.

Reviewer #3 (Recommendations For The Authors):

Page 6, Figure 2, Page 23 (Methods)

"The supernatants were purified with a Ni-Agarose resin column followed by size-exclusion chromatography."

What kind of SEC column did the authors use? Can the authors provide the SEC elution profile comparison results and size standard curve?

Response: We use a Superdex 200 (Hiload 16/600) column for size exclusion chromatography. The comparison results of SEC elution profiles for AtSerDC and CsAlaDC, along with the standard curve of SEC column, are presented below.

Author response image 2.

(A) Comparison of elution profiles of CsAlaDC and AtSerDC. (B) Elution profile of Blue Dextron 2000. (C) Elution profile of mixed protein (Aldolase, 158000 Da,71.765ml; Conalbumin, 75000 Da,79.391ml; Ovalbumin, 44000 Da,83.767ml; Carbonic anhydrase, 29000 Da,90.019ml; Ribonuclease A, 13700 Da,98.145ml). (D) Size standard curves of Superdex 200 (Hiload 16/600) column.

Page 6 & Page 24 (Methods)

"The 100 μL reaction mixture, containing 20 mM substrate (Ala or Ser), 100 mM potassium phosphate, 0.1 mM PLP, and 0.025 mM purified enzyme, was prepared and incubated at standard conditions (45 ℃ and pH 8.0 for CsAlaDC, 40 ℃ and pH 8.0 for AtSerDC for 30 min)."

(1) The enzymatic activities of CsAldDC and AtSerDC were measured at two different temperatures (45 and 40 ℃, but their activities were directly compared. Is there a reason for experimenting at different temperatures?

Response: We determined that the optimal reaction temperature for AtSerDC is 40°C and for CsAlaDC is 45°C through our verification process. Consequently, all subsequent experiments were performed at these specific temperatures.

Author response image 3.

(A) Relative activity of CsAlaDC at different temperatures. (B) Relative activity of AtSerDC at different temperatures.

(2) Enzyme activities were measured at temperatures above 40℃, which is not a physiologically relevant temperature and may affect the stability or activity of the proteins. At the very least, the authors should provide temperature-dependent protein stability data (e.g., CD spectra analysis) or, if possible, temperature-dependent enzyme activities, to show that their experimental conditions are suitable for studying the activities of these enzymes.

Response: Thank you very much for your careful reading. We have already validated that the experimental temperature we used did not significantly affect the stability of the protein before experimenting. The results are shown in the figure below:

Author response image 4.

Place the two proteins individually into water baths set at temperatures of 25°C, 37°C, 45°C, 60°C, and 80°C for 15 minutes. Subsequently, carry out enzymatic reactions utilizing a standard reaction system, with untreated enzymes serving as the experimental control within the said system. The experimental results suggest that the temperature at which we experimented does not have a significant impact on the stability of the enzyme.

(3) The authors used 20 mM of substrate. What are the physiological concentrations of alanine and serine typically found in plants?

Response: The content of alanine in tea plant roots ranges from 0.28 to 4.18 mg/g DW (Yu et al., 2021; Cheng et al., 2017). Correspondingly, the physiological concentration of alanine is 3.14 mM to 46.92 mM, in tea plant roots. The content of serine in plants ranges from 0.014 to 17.6 mg/g DW (Kumar et al., 2017). Correspondingly, the physiological concentration of serine is 0.13 mM to 167.48 mM in plants. In this study, the substrate concentration of 20 mM was close to the actual concentrations of alanine and serine in plants.

Yu, Y. et al. (2021) Glutamine synthetases play a vital role in high accumulation of theanine in tender shoots of albino tea germplasm "Huabai 1". J. Agric. Food Chem. 69 (46),13904-13915.

Cheng, S. et al. (2017) Studies on the biochemical formation pathway of the amino acid L-theanine in tea (Camellia sinensis) and other plants.” J. Agric. Food Chem. 65 (33), 7210-7216.

Kumar, V. et al. (2017) Differential distribution of amino acids in plants. Amino Acids. 49(5), 821-869.

Pages 6-7 & Table 1

(1) Use the correct notation for Km and Vmax. Also, the authors show kinetic parameters and use multiple units (e.g., mmol/L or mM for Km).

Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected this in the revised manuscript.

(2) When comparing the catalytic efficiency of enzymes, kcat/Km (or Vmax/Km) is generally used. The authors present a comparison of catalytic activity from results to conclusion. A clarification of what results are being compared is needed.

Response: Thank you for your comments and suggestions. The catalytic activity is assessed by comparing reaction rates.

Page 7 & Figure 3

In Figure 3A, the authors describe the overall structure, but a simple explanation or labeling within the figure should be added.

Response: Thank you very much for your suggestions, we have made modifications to Figure 3A as follows:

Author response image 5.

Crystal structures of CsAlaDC and AtSerDC. (A) Dimer structure of CsAlaDC. The color display of the N-terminal domain, large domain, and C-terminal domains of chain A is shown in light pink, khaki and sky blue, respectively. Chain B is shown in spring green. The PLP molecule is shown as a sphere model. The zinc finger structure at the C-terminus of CsAlaDC is indicated by the red box. The gray spheres represent zinc ions, while the red dotted line depicts the coordination bonds formed by zinc ions with cysteine and histidine.

Figures 3F & 4A

In these figures, the two structures are overlaid and compared, but the colors are very similar to see the differences. The authors should use a different color scheme.

Response: Thank you very much for your suggestions, we have made modifications to the Figure 3F & 4A as follows:

Author response image 6.

(Figure 3F) The monomers of CsAlaDC and AtSerDC are superimposed. CsAlaDC is depicted in spring green, while AtSerDC is shown in plum. The conserved amino acid catalytic ring is indicated by the red box.

(Figure 4A) Superposition of substrate binding pocket amino acid residues in CsAlaDC and AtSerDC. The amino acid residues of CsAlaDC are shown in spring green, the amino acid residues of AtSerDC are shown in plum, with the substrate specificity-related amino acid residue highlighted in a red ellipse.

Pages 7 & 8

Figures 3 and 4 do not include illustrations of what the authors describe in the text. The reader will not be able to understand the descriptions until they download and view the structures themselves. The authors should create additional figures to make it easier for readers to understand the structures.

Response: Thank you very much for your suggestions, we have included supplementary figure 1 in the revised manuscript, which presents more elaborate structural depictions of the two proteins.

Pages 9 & 10

"This result suggested this Tyr is required for the catalytic activity of CsAlaDC and AtSerDC."

The author's results are interesting, but it is recommended to perform the experiments in a specific order. First, experiments should determine whether mutagenesis affects the protein's stability (e.g., CD, as discussed earlier), and second, whether mutagenesis affects ligand binding (e.g., ITC, SPR, etc.), before describing how site-directed mutagenesis alters enzyme activity. In particular, the authors' hypothesis would be much more convincing if they could show that the ligand binding affinity is similar between WT and mutants.

Response: Thank you for your insightful feedback on our manuscript, which we greatly appreciate. Your suggestion to methodically sequence the experiments provides a clear pathway to bolster the strength and conclusiveness of our results.

We agree that it is crucial to first assess the stability of the mutant proteins, as changes therein could inadvertently affect catalytic activity. To this end, we have employed circular dichroism (CD) to study the potential structural alterations in the proteins induced by mutations. The experimental results are shown in the following figure:

Author response image 7.

(A) Circular Dichroism Spectra of CsAlaDC (WT). (B) Circular Dichroism Spectra of CsAlaDC (Y336F). (C) Circular Dichroism Spectra of CD of AtSerDC (WT). (D) Circular Dichroism Spectra of AtSerDC (Y341F).

The experimental results indicate that the secondary structure of the mutant proteins remains unchanged, which means the mutations do not alter the protein's stability.

The ligand PLP forms a Schiff base structure with the ε-amino group of a lysine residue in the protein, with maximum absorbance around 420-430 nm. Since we have already added PLP during the protein purification process, as long as the absorbance of mutant proteins and wild-type proteins is the same at 420-430 nm at equivalent concentrations, it indicates that the mutant proteins do not affect the binding of the ligand PLP. Therefore, we scanned the UV-visible absorption spectra of both the wild-type and mutant proteins, and the results are as presented in the following figure:

Author response image 8.

(A) UV-Visible Absorption Spectra of CsAlaDC (WT) compared to CsAlaDC (Y336F). (B) UV-Visible Absorption Spectra of AtSerDC (WT) compared to AtSerDC (Y341F).

The mutant protein and the wild-type protein exhibit similar absorbance at 420-430 nm, indicating that the mutation does not affect the binding of PLP to the protein.

The above experiments have confirmed that the mutations do not significantly affect the stability of the protein or the affinity for the ligand, so we can more confidently attribute changes in enzyme activity to the specific role of the tyrosine residue in question. We believe this comprehensive approach will substantiate our hypothesis and illustrate the necessity of this Tyr residue for the catalytic activity of CsAlaDC and AtSerDC enzymes.

Figure 3

In the 3D structure figure provided by the authors, the proposed reaction mechanism of the enzyme and the involved amino acids are not included. Can the authors add a supplementary figure with a schematic drawing that includes more information, such as distances?

Response: Thank you for your valuable feedback on our manuscript. We completely agree that a schematic drawing with additional details, including distances, would enhance the clarity and understanding of the enzymatic mechanism. In response to your suggestion, we have added a supplementary figure 2 in the revised manuscript that accurately illustrates the proposed reaction pathway, highlighting the key amino acids involved.

Page 10

"The results showed that 5 mM L-DTT reduced the relative activity of CsAlaDC and AtSerDC to 22.0% and 35.2%, respectively"

The authors primarily use relative activity to compare WT and mutants. Can the authors specify the exact experiments, units, and experimental conditions? Is it Vmax or catalytic efficiency? If so, under what specific experimental conditions?

Response: Thank you for your attention and review of our research paper, we appreciate your suggestions and feedback. The experimental protocol employed to evaluate the influence of DTT on protein catalytic efficiency is outlined as follows:

The 100 μL reaction mixture, containing 20 mM substrate (Ala or Ser), 100 mM potassium phosphate, 0.1 mM PLP, 5 mM L-DTT, and 0.025 mM purified enzyme, was prepared and incubated at standard conditions (45 °C and pH 8.0 for CsAlaDC for 5 min, 40 °C and pH 8.0 for AtSerDC for 2 min). DTT is absent as a control in the reaction system. Then the reaction was stopped with 20 μL of 10% trichloroacetic acid. The product was derivatized with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) and subjected to analysis by UPLC. All enzymatic assays were performed in triplicate.

However, due to the unknown mechanism of DTT inhibition on protein activity, we have removed this part of the content in the revised manuscript.

Pages 10-12

The identification of 'Phe106 in CsAlaDC' and 'Tyr111 in AtSerDC,' along with the subsequent mutagenesis and enzymatic activity assays, is intriguing. However, the current manuscript lacks an explanation and discussion of the underlying reasons for these results. As previously mentioned, it would be helpful to gain insights and analysis from WT-ligand and mutant-ligand binding studies (e.g., ITC, SPR, etc.). Furthermore, the authors' analysis would be more convincing with accompanying structural analysis, such as steric hindrance analysis.

Response: Thank you for your insightful comments and constructive feedback on our manuscript. We appreciate the interest you have expressed in the identification of 'Phe106 in CsAlaDC' and 'Tyr111 in AtSerDC' and their functional implications based on mutagenesis and enzymatic assays.

In order to investigate the binding status of the mutant protein and the ligand PLP,we scanned the UV-visible absorption spectra of both the wild-type and mutant proteins, and the results are as presented in the following figure:

Author response image 9.

(A) UV-Visible Absorption Spectra of CsAlaDC (WT) compared to CsAlaDC (F106Y). (B) UV-Visible Absorption Spectra of AtSerDC (WT) compared to AtSerDC (Y111F).

The mutant protein and the wild-type protein exhibit similar absorbance at 420-430 nm, indicating that the mutation does not affect the binding of PLP to the protein. Therefore, we can conclude that the change in activity of the mutant protein is caused by the substitution of the amino acid at that site, i.e., the amino acid at that site affects substrate specificity. By combining the structure of the two proteins, we can see that the Lys at position 111 of AtSerDC is a hydrophilic amino acid, which increases the hydrophilicity of the active site, and thus the substrate is the hydrophilic amino acid Ser. In contrast, the amino acid at the corresponding site in CsAlaDC is Phe, which, lacking a hydroxyl group compared to Lys, increases the hydrophobicity of the active site, making the substrate lean towards the hydrophobic amino acid Ala. We have added a discussion of the potential reasons for this result to the revised manuscript's discussion section.

Page 5 & Figure 1B

"As expected, CsSerDC was most closed to AtSerDC, which implies that they shared similar functions. However, CsAlaDC is relatively distant from CsSerDC."

In Figure 1B, CsSerDC and AtSerDC are in different clades, and this figure does not show that the two enzymes are closest. To provide another quantitative comparison, please provide a matrix table showing amino acid sequence similarities as a supplemental table.

Response: Many thanks for your constructive suggestion. We added a matrix table showing amino acid sequence similarities in the supplemental materials. The results showed that the similarity of amino acid sequences between CsSerDC and AtSerDC is 86.21%, which is higher than that between CsAlaDC and CsSerDC (84.92%). This data exactly supports the description of Figure 1B. We added the description of the amino acid sequence similarities analysis in the revised manuscript. The description of "As expected, CsSerDC was most closed to AtSerDC, which implies that they shared similar functions. " is not accurate enough, so we revised it to "As expected, CsSerDC was closer to AtSerDC, which implies that they shared similar functions.", in the revised manuscript.

Page 5 & Figure 1C

Figure 1C, which shows a multiple sequence alignment with the amino acid sequences of the 6 SerDCs and CsAlaDC, clearly shows the differences between the sequences of AlaDC and other SerDCs. However, the authors' hypothesis would be more convincing if they showed that this difference is also conserved in AlaDCs from other plants. Can the authors show a new multiple-sequence alignment by adding more amino acid sequences of other AlaDCs?

Response: Thank you for your comments and suggestions. We aim to discover additional alanine decarboxylase. However, at present, the only experimentally confirmed alanine decarboxylase is CsAlaDC. No experimentally verified alanine decarboxylases have been found in other plant species.

Figure 5A

Figure 5A is missing the error bar.

Response: Figure 5A serves as a preliminary screening for these mutants, without conducting repeated experiments. Subsequently, only the L110F and P114A mutants, which exhibited significantly improved activity, underwent further experimental verification to confirm their enhanced functionality.

Author Response

The following is the authors’ response to the original reviews.

Thank you for your time and effort in handling and reviewing our manuscript. We have responded to all comments below.

Reviewer #1 (Public Review):

Martinez-Gutierrez and colleagues presented a timeline of important bacteria and archaea groups in the ocean and based on this they correlated the emergence of these microbes with GOE and NOE, the two most important geological events leading to the oxygen accumulation of the Earth. The whole study builds on molecular clock analysis, but unfortunately, the clock analysis contains important errors in the calibration information the study used, and is also oversimplified, leaving many alternative parameters that are known to affect the posterior age estimates untested. Therefore, the main conclusion that the oxygen availability and redox state of the ocean is the main driver of marine microbial diversification is not convincing.

We do not conclude that “oxygen availability and redox state of the ocean is the main driver of marine microbial diversification”. Our conclusion is much more nuanced. We merely discuss our findings in light of the major oxygenation events and oxygen availability (among other things) given the important role this molecule has played in shaping the redox state of the ocean.

Regarding the methodological concerns, to address them we have provided additional analyses to account for different clock models and calibration points.

Basically, what the molecular clock does is to propagate the temporal information of the nodes with time calibrations to the remaining nodes of the phylogenetic tree. So, the first and the most important step is to set the time constraints appropriately. But four of the six calibrations used in this study are debatable and even wrong.

(1) The record for biogenic methane at 3460 Ma is not reliable. The authors cited Ueno et al. 2006, but that study was based on carbon isotope, which is insufficient to demonstrate biogenicity, as mentioned by Alleon and Summons 2019.

Thank you for pointing out the limitations of using the geochemical evidence of methane as calibrations. Indeed, several commentaries have suggested that the biotic and abiotic origin of the methane reported by Ueno et al. are equally plausible (Alleon and Summons, 2019; Lollar and McCollom, 2006), however; we used that calibration as a minimum for the presence of life on Earth, not methanogenesis. Despite the controversy regarding the origin of methane, there are other lines of evidence suggesting the presence of life around ~3.4 Ga. For example stromatolites from the Dresser Formation, Pilbara, Western Australia (Djokic et al., 2017; Walter et al., 1980; Buick and Dunlop, 1990), and more recently (Hickman-Lewis et al., 2022). To avoid confusion, we have added a more extended explanation for the use of that calibration and additional evidence of life around that time in Table 1 and lines 100-104.

(2) Three calibrations at Aerobic Nitrososphaerales, Aerobic Marinimicrobia, and Nitrite oxidizing bacteria have the same problem - they are all assumed to have evolved after the GOE where the Earth started to accumulate oxygen in the atmosphere, so they were all capped at 2320 Ma. This is an important mistake and will significantly affect the age estimates because maximum constraint was used (maximum constraint has a much greater effect on age estimates and minimum constraint), and this was used in three nodes involving both Bacteria and Archaea. The main problem is that the authors ignored the numerous evidence showing that oxygen can be produced far before GOE by degradation of abiotically-produced abundant H2O2 by catalases equipped in many anaerobes, also produced by oxygenic cyanobacteria evolved at least 500 Ma earlier than the onset of GOE (2500 Ma), and even accumulated locally (oxygen oasis). It is well possible that aerobic microbes could have evolved in the Archaean.

We appreciate the suggestion of assessing the validity of the calibrations used in our analyses. We initially evaluated the informative power of the priors used for the Bayesian molecular dating (Supplemental File 5), and found that the only calibration that lacked enough information for the purposes of our study was Ammonia Oxidizing Archaea (AOA). In contrast to previous evidence (Ren et al., 2019; Yang et al., 2021), we associate this finding to the potential earlier diversification of AOA. Due to the limitations of several of the calibrations used, we performed an additional molecular dating analysis on 1000 replicate trees using a Penalized Likelihood strategy. This analysis consisted in excluding the calibrations that assumed the presence of oxygen as a maximum constraint. Our analysis shows similar age estimates of the marine microbial clades regardless of the exclusion of these calibrations (Supplemental File 8; TreePL Priors set 2). Our findings thus suggest that the age estimates reported in our study are consistent regardless of whether or not the presence of oxygen is used to calibrate several nodes in the tree. We describe the results of this analysis in lines 490-499 and include estimates in Supplemental File 8. Our results are therefore robust regardless of the use of these somewhat controversial calibrations.

Once the phylogenetic tree is appropriately calibrated with fossils and other time constraints, the next important step is to test different clock models and other factors that are known to significantly affect the posterior age estimates. For example, different genes vary in evolutionary history and evolutionary rate, which often give very different age estimates. So it is very important to demonstrate that these concerns are taken into account. These are done in many careful molecular dating studies but missing in this study.

We agree that the selection of marker genes will have a profound impact on the final age estimates. First, it is important to understand that very few genes present in modern Bacteria and Archaea can be traced back to the Last Universal Common Ancestor, so there are very few genes to use for this purpose. Studies that focus on particular groups of Bacteria and Archaea may have larger selections of genes to choose from, but for our purposes there are only about ~40 different genes - mostly encoding for ribosomal proteins, RNA polymerase subunits, and tRNA synthetases - that can be use for this purpose (Creevey et al., 2011; Wu and Scott, 2012). In a previous study we have extensively benchmarked methods for the reconstruction of high-resolution phylogenetic trees of Bacteria and Archaea using these genes (Martinez-Gutierrez and Aylward, 2021). Our analyses demonstrated that some of these genes (mainly tRNA synthetases) have undergone ancient lateral gene transfer events and are not suitable for deep phylogenetics or molecular dating. In this previous study we also evaluated different sets of marker genes to examine which provide the most robust phylogenetic inference. We arrived at a set of ribosomal proteins and RNA polymerase subunits that performs best for phylogenetic reconstruction, and we have used that in the current study.

Furthermore, we tested the role of molecular dating model selection on the final Bayesian estimates by running four independent chains under the models UGAM and CIR, respectively. Overall, the results did not vary substantially compared with the ages obtained using the log-normal model reported on our manuscript (Supplemental File 8). The additional results are described in lines 478-488 and shown in Supplemental File 8. The clades that showed more variation when using different Bayesian models were SAR86, SAR11, and Crown Cyanobacteria (Supplemental File 8). Despite observing some differences in the age estimates when using different molecular models, the conclusion that the different marine microbial clades presented in our study diversified during distinct periods of Earth’s history remains. Moreover, the main goal of our study is to provide a relative timeline of the diversification of abundant marine microbial clades without focusing on absolute dates.

Reviewer #2 (Public Review):

In this paper, Martinez-Gutierrez and colleagues present a dated, multidomain (= Archaea+Bacteria) phylogenetic tree, and use their analyses to directly compare the ages of various marine prokaryotic groups. They also perform ancestral gene content reconstruction using stochastic mapping to determine when particular types of genes evolved in marine groups.

Overall, there are not very many papers that attempt to infer a dated tree of all prokaryotes, and this is a distinctive and up-to-date new contribution to that oeuvre. There are several particularly novel and interesting aspects - for example, using the GOE as a (soft) maximum age for certain groups of strictly aerobic Bacteria, and using gene content enrichment to try to understand why and how particular marine groups radiated.

Thank you for your thorough evaluation and comments on our manuscript.

Comments

One overall feature of the results is that marine groups tend to be quite young, and there don't seem to be any modern marine groups that were in the ocean prior to the GOE. It might be interesting to study the evolution of the marine phenotype itself over time; presumably some of the earlier branches were marine? What was the criterion for picking out the major groups being discussed in the paper? My (limited) understanding is that the earliest prokaryotes, potentially including LUCA, LBCA and LACA, was likely marine, in the sense that there would not yet have been any land above sea level at such times. This might merit discussion in the paper. Might there have been earlier exclusively marine groups that went extinct at some point?

Thank you for pointing this out - this is a very interesting idea.<br /> Firstly, the major marine lineages that we study here have largely already been defined in previous studies and are known to account for a large fraction of the total diversity and biomass of prokaryotes in the ocean. For example, Giovannoni and Stingl described most of these groups previously when discussing cosmopolitan and abundant marine lineages (Giovannoni and Stingl, 2005). The main criteria to select the marine clades studied here are 1) these groups have large impacts in the marine biogeochemical cycles and represent a large fraction of the microbial biomass in the open ocean, 2) they have an appropriate representation on genomic databases such that they can be confidently included in a phylogenetic tree, 3) the clades included can be confidently classified as being marine, in the sense that consequently the last common ancestor had a marine origin. This is explained in lines 83-86. We were primarily interested in lineages that encompassed a broad phylogenetic breadth, and we therefore did not include many groups that can be found in the ocean but are also readily isolated from a range of other environments (i.e., Pseudomonas spp., some Actinomycetes, etc.).

We agree that some of the earlier microbial branches in the Tree of Life were likely marine. The study of the marine origin of LUCA, LBCA, LACA, although interesting, is out of the scope of our study, and our results cannot offer any direct evidence of their habitat. We have therefore sought to focus on the origins of extant marine lineages.

What do the stochastic mapping analyses indicate about the respective ancestors of Gracilicutes and Terrabacteria? At least in the latter case, the original hypothesis for the group was that they possessed adaptations to life on land - which seems connected/relevant to the idea of radiating into the sea discussed here - so it might be interesting to discuss what your analyses say about that idea.

Thank you for your recommendation to perform additional analysis regarding the characterization of the ancestor of the superphyla Gracilicutes and Terrabacteria. We agree that this analysis would be very interesting, but we wish to focus the manuscript primarily on the marine clades in question, and other supergroups are listed in Figure 2 mainly for context. However, we did check the results of the stochastic mapping analysis and we now report the list of genes predicted to be gained and lost at the ancestor of the Gracilicutes and Terrabacteria clades, however; it is out of the scope of this study.

I very much appreciate that finding time calibrations for microbes is challenging, but I nonetheless have a couple of comments or concerns about the calibrations used here:

The minimum age for LBCA and LACA (Nodes 1 and 2 in Fig. 1) was calibrated with the earliest evidence of biogenic methane ~3.4Ga. In the case of LACA, I suppose this reflects the view that LACA was a methanogen, which is certainly plausible although perhaps not established with certainty. However, I'm less clear about the logic of calibrating the minimum age of Bacteria using this evidence, as I am not aware that there is much evidence that LBCA was a methanogen. Perhaps the line of reasoning here could be stated more explicitly. An alternative, slightly younger minimum age for Bacteria could perhaps be obtained from isotope data ~3.2Ga consistent with Cyanobacteria (e.g., see https://pubmed.ncbi.nlm.nih.gov/30127539/).

Thank you for pointing this out. We used the presence of methane as a minimum for life on Earth, not as a minimum for methanogenesis. Despite using this calibration as a minimum for the root of Bacteria and not having methanogenic representatives within this domain, there are independent lines of evidence that point to the presence of microbial life around the same time (~3.5 Ga, for example stromatolites from the Dresser Formation, Pilbara, Western Australia (~3.5 Ga) (Djokic et al., 2017; Walter et al., 1980; Buick and Dunlop, 1990), and more recently (Hickman-Lewis et al., 2022). We added a rationale for the use of the evidence of methane as a minimum age for life on Earth to the manuscript (Table 1 and 100104).

I am also unclear about the rationale for setting the minimum age of the photosynthetic Cyanobacteria crown to the time of the GOE. Presumably, oxygen-generating photosynthesis evolved on the stem of (photosynthetic) Cyanobacteria, and it therefore seems possible that the GOE might have been initiated by these stem Cyanobacteria, with the crown radiating later? My confusion here might be a comprehension error on my part - it is possible that in fact one node "deeper" than the crown was being calibrated here, which was not entirely clear to me from Figure 1. Perhaps mapping the node numbers directly to the node, rather than a connected branch, would help? (I am assuming, based on nodes 1 and 2, that the labels are being placed on the branch directly antecedent to the node of interest)?

Thank you so much for your suggestion. As pointed out, the calibrations used were applied at the crown node of existing Cyanobacterial clades, not at the stem of photosynthetic Cyanobacteria. We agree that photosynthesis and therefore the production of molecular oxygen may have been present in more ancient Cyanobacterial clades, however; these groups have not been discovered yet or went extinct. We have improved Fig. 1 to avoid confusion and now it is part of the updated version of our manuscript.

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Author Response

The following is the authors’ response to the original reviews.

We thank the editor and the reviewers for their very useful and constructive comments. We went through the list and gladly received all their suggestions. The reviewers mostly pointed to minor revisions in the text, and we acted on all of those. The one suggestion that required major work was the one raised in point 13, about the processing pipeline being unconvincingly scattered between different tools (R → Python → Matlab). I agree that this was a major annoyance, and I am happy to say we have solved it integrating everything in a recent version of the ethoscopy software (available on biorxiv with DOI https://www.biorxiv.org/content/10.1101/2022.11.28.517675v2 and in press with Bioinformatics Advances). End users will now be able to perform coccinella analysis using ethoscopy only, thus relying on nothing else but Python as their data analysis tool. This revised version of the manuscript now includes two Jupyter Notebooks as supplementary material with a “pre-cooked” sample recipe of how to do that. This should really simplify adoption and provides more details on the pipeline used for phenotyping.

Please find below a point-by-point description of how we incorporated all the reviewers’ excellent suggestions.

Recommendations for the authors: please note that you control which, if any, revisions, to undertake

1) Line 38: "collecting data simultaneously from a large number of individuals with no or limited human intervention" is a bit misleading, as the entire condition the individuals are put in are highly modified by humans and most times "unnatural". I understand the point that once the animals are placed in these environments, then recording takes place without intervention, but it would be nice to rephrase this so that it reflects more accurately what is happening.

We have now rephrased this into the following (L39):

Collecting data simultaneously from a large number of individuals, which can remain undisturbed throughout recording.

2) Line 63: please add a reference to the Ethoscopes so that readers can easily find it.

Done.

2b) And also add how much they cost and the time needed to build them, as this will allow readers to better compare the proposed system against other commercially available ones.

This information is available on the ethoscope manual website (http://lab.gilest.ro/ethoscope). The price of one ethoscope, provided all necessary tools are available, is around ~£75 and the building time very much depends on the skillset of the builder and whether they are building their first ethoscope or subsequent ones. In our experience, building and adopting ethoscopes for the first time is not any more time-expensive than building a (e.g.) deeplabcut setup for the first time. We have added this information to L81

Ethoscopes are open source and can be manufactured by a skilled end-user at a cost of about £75 per machine, mostly building on two off-the-shelf component: a Raspberry Pi microcomputer and a Raspberry Pi NoIR camera overlooking a bespoke 3D printed arena hosting freely moving flies.

3) Line 88: The authors describe that in the current setting, their system is capable of an acquisition rate of 2.2 frames per second (FPS). Would reducing the resolution of the PiCamera allow for higher FPS? I raise this point because the authors state that max velocity over a ten second window is a good feature for classifying behaviors. However, if animals move much faster than the current acquisition rate, they could, for instance, be in position X, move about and be close to the initial position when the next data point is acquired, leading to a measured low max velocity, when in fact the opposite happened. I think it would be good to add a statement addressing this (either data from the literature showing that the low FPS does not compromise data acquisition, or a test where increasing greatly FPS leads to the same results).

We have previously performed a comparison of data analysed using videos captured at different FPSs, which is published in Quentin Geissman’s doctoral Thesis (2018, DOI: https://doi.org/10.25560/69514 ) in chapter 2, section 2.8.3, figure 2.9 ). We have now added this work as one of the references at L95 (reference 19).

4) Still on the low FPS, would a Raspberry Pi 4 help with the sampling rate? Given that they are more powerful than the RPi3 used in the paper?

It would, but it would be a minor increase, leading from 2.2 to probably 3-5 FPS. A significantly higher number of FPSs would be best achieved by lowering the camera’s resolution, as the reviewer’s suggested, or by operating offline. I think the interesting point being implied by the reviewers is that, for Drosophila, the current limits of resolution are more than sufficient. For other animals, perhaps moving more abruptly, they may not. The reviewer is right that we should add a line of caveat about this. We now do so in the discussion, lines 215-224.

Coccinella is a reductionist tool, not meant to replace the behavioural categorization that other tools can offer but to complement it. It relies on raspberry PIs as main acquisition devices, with associated advantages and limitations. Ethoscopes are inexpensive and versatile but have limitations in terms of computing power and acquisition rates. Their online acquisition speed is fast enough to successfully capture the motor activity of different species of Drosophilae28, but may not be sufficient for other animals moving more swiftly, such as zebrafish larvae. Moreover, coccinella cannot apply labels to behaviour (“courting”, “lounging”, “sipping”, “jumping” etc.) but it can successfully identify large behavioural phenotypes and generate unbiased hypothesis on how behaviour – and a nervous system at large – can be influenced by chemicals, genetics, artificial manipulations in general.

5) Along the same line of thought, would using a simple webcam (with similar specs to the PiCamera - ELP has cameras that operate on infrared and are quite affordable too) connected to a more powerful computer lead to higher FPS? - The reason for the question about using a simple webcam is that this would make your system more flexible (especially useful in the current shortage of RPi boards on the market) lowering the barrier for others to use it, increasing the chances for adoption.

Completely bypassing ethoscopes would require the users to setup their own tracking solution, with a final result that may or may not match what we describe here. If a greater temporal resolution is necessary, the easiest way to achieve more FPSs would be to either decrease camera resolution or use the Pis to take videos offline and then process those videos at a later stage. The combination of these two would give FPS acquisition of 60 fps at 720p, which is the maximum the camera can achieve. We now made this clear at lines 83-92.

The temporal and spatial resolution of the collected images depends on the working modality the user chooses. When operating in offline mode, ethoscopes are capable to acquire 720p videos at 60 fps, which is a convenient option with fast moving animals. In this study, we instead opted for the default ethoscope working settings, providing online tracking and realtime parametric extraction, meaning that images are analysed by each raspberry Pi at the very moment they were acquired (Figure 1b). This latter modality limits the temporal resolution of information being processed (one frame every 444 ms ± 127 ms, equivalent to 2.2 fps on a Raspberry Pi3 at a resolution of 1280x960 pixels with each animal being constricted in an ellipse measuring 25.8 ± 1.4 x 9.85 ±1.4 pixels - Figure 1a) but provides the most affordable and high-throughput solution, dispensing the researcher from organising video storage or asynchronous video processing for animals tracking.

6) One last point about decreasing use barrier and increasing adoption: Would it be possible to use DeepLabCut (DLC) to simply annotate each animal (instead of each body part) and feed the extracted data into your current analysis with coccinella? This way different labs that already have pipelines in place that use DLC would have a much easier time in testing and eventually switching to coccinella? I understand that extracting simple maximal velocity this way would be an overkill, but the trade-off would again be a lowering of the adoption barrier.

It would certainly be possible to calculate velocity from the whole animal pose measurement and then use this with HCTSA or Catch22, thus mimicking the coccinella pipeline, but it would be definitely overkilled, as the reviewers correctly points out. Given that we are trying to make an argument about high-throughput data acquisition I would rather not suggest this option in the manuscript.

7) Line 96: The authors state that once data is collected, it is put through a computational frameworkthat uses 7700 tests described in the literature so that meaningful discriminative features are found. I think it would be interesting to expand a bit on the explanation of how this framework deals multiple comparison/multiple testing issues.

We always use the full set of features on aggregate to train a classifier (e.g., TS_Classify in HCTSA) and that means no correction is necessary because the trained classifier only ever makes a single prediction (only one test is performed), so as long as it is done correctly (e.g., proper separation of training and test sets, etc.) then multiple hypothesis correction is not appropriate. This has been confirmed with the HCTSA/Catch22 author (Dr Ben Fulcher, personal communication). We have added a clarifying sentence about this to the methods (L315-318)

8) It would be nice to have a couple of lines explaining the choice of compounds used for testing and also why in some tests, 17 compounds were used, while in others 40, and then 12? I understand how much work it must be in terms of experiment preparation and data collection for these many flies and compounds, but these changes in the compounds used for testing without a more detailed explanation is suboptimal.

This is another good point. We have now added this information to the methods, in a section renamed “choice, handling and preparation of drugs” L280-285, which now reads like this:

The initial preliminary analysis was conducted using a group of 12 compounds “proof of principle” compounds and a solvent control. These compounds were initially used to compare both the video method and ethoscope method. After testing these initial compounds, it was found that the ethoscope methodology was more successful, and then the compound list was expanded to 17 (including the control) only using the ethoscope method. As a final test, we included additional compounds for a single concentration, bringing up the total to 40 (including control), also for the ethoscope method.

9) Line 119 states: "A similar drop in accuracy was observed using a smaller panel of 12 treatments (Supplementary Figure 2a)". It is actually Supplementary Figure 1c.

Thank you for noticing that! Now corrected. The Supplementary figures have also been renamed to obey eLife’s expected nomenclature (both Figure 1 – Figure supplements)

10) In some places the language seems a little outlandish and should either be removed or appropriately qualified. a- Lines 56-59 pose three questions that are either rhetorical or ill-posed. For example, "...minimal amount of information...behavior" implies there is a singular response but the response depends on many details such as to what degree do the authors want to "classify behavior".

Yes, those were meant as rhetorical questions indeed, but we prefer to keep them in, because we are hoping to generate this type of thoughts with the readers. These are concepts that may not be so obvious to someone who is just looking to apply an existing tool and may spring some reflection about what kind of data do they really want/need to acquire.

b) Some of the criticisms leveled at the state-of-the-art methods are probably unwarranted because the goals of the different approaches are different. The current method does not yield the type of rich information that DeepLabCut yields. So, depending on the application DeepLabCut may be the method of choice. The authors of the current manuscript should more clearly state that.

In the introduction and discussion we do try to stress that coccinella is not meant to replace tools like DLC. We have now added more emphasis to this concept, for instance to L212:

[tools like deeplabcut] are ideal – and irreplaceable – to identify behavioural patterns and study fine motor control but may be undue for many other uses.

And L215:

Coccinella is a reductionist tool not meant to replace the behavioural categorization that other tools can offer but to complement it

11) The application to sleep data appears suddenly in the manuscript. The authors should attempt to make with text change a smoother transition from drug screen to investigation into sleep.

I agree with this observation. We have now tried to add a couple of sentences to contextualise this experiment and hopefully make the connection appear more natural. Ultimately, this is a proof-ofprinciple example anyway so hopefully the reader will take it for what it is (L169).

Finally, to push the system to its limit, we asked coccinella to find qualitative differences not in pharmacologically induced changes in activity, but in a type of spontaneous behaviour mostly characterised by lack of movement: sleep. In particular, we wondered whether coccinella could provide biological insights comparing conditions of sleep rebound observed after different regimes of sleep deprivation. Drosophila melanogaster is known to show a strong, conserved homeostatic regulation of sleep that forces flies to recover at least in part lost sleep, for instance after a night of forceful sleep deprivation.

11b) Additionally, the beginning section of sleep experiments talks about sleep depth yet the conclusion drawn from sleep rebound says more about the validity of the current 5 min definition of sleep than about sleep depth. If this conclusion was misunderstood, it should be clarified. If it was not, the beginning text of the sleep section should be tailored to better fit the conclusion.

I am afraid we did not a good job at explaining a critical aspect here: the data fed to coccinella are the “raw” activity data, in which we are not making any assumption on the state of the animal. In other words, we do not use the 5-minutes at this or any other point to classify sleep and wakening. Nevertheless, coccinella picks the 300 seconds threshold as the critical one for discerning the two groups. This is interesting because it provides a full agnostic confirmation of the five minutes rule in D. melanogaster. We recognise this was not necessarily obvious from the text and now added a clarification at L189-201:

However, analysis of those same animals during rebound after sleep deprivation showed a clear clustering, segregating the samples in two subsets with separation around the 300 seconds inactivity trigger (Figure 3d). This result is important for two reasons: on one hand, it provides, for the third time, strong evidence that the system is not simply overfitting data of nought biological significance, given that it could not perform any better than a random classifier on the baseline control. On the other hand, coccinella could find biologically relevant differences on rebound data after different regimes of sleep deprivation. Interestingly enough, the 300 seconds threshold that coccinella independently identified has a deep intrinsic significance for the field, for it is considered to be the threshold beyond which flies lose arousal response to external stimuli, defining a “sleep quantum” (i.e.: the minimum amount of time required for transforming inactivity bouts into sleep bouts23,24,28). Coccinella’s analysis ran agnostic of the arbitrary 5-minutes threshold and yet identified the same value as the one able to segregate the two clusters, thus providing an independent confirmation of the fiveminutes rule in D. melanogaster.

12) Line 227: (standard food) - please add a link to a protocol or a detailed description on what is "standard food". This way others can precisely replicate what you are using. This is not my field, but I have the impression that food content/composition for these animals makes big changes in behaviour?

Yes, good point. We have now added the actual recipe to the methods L240:

Fly lines were maintained on a 12-hour light: 12-hour dark (LD) cycle and raised on polenta and yeast-based fly media (agar 96 g, polenta 240 g, fructose 960 g and Brewer’s yeast 1,200 g in 12 litres of water).

13) Data acquisition and processing: please add links to the code used.

Both the code and the raw data used to generate all the figures have been uploaded on Zenodo and available through their repository. Zenodo has a limit of 50GB per uploaded dataset so we had to split everything into two files, with two DOIs, given in the methods (L356, section “code and availability” - DOIs: 10.5281/zenodo.7335575 and 10.5281/zenodo.7393689). We have now also created a landing page for the entire project at http://lab.gilest.ro/coccinella and linked that landing page in the introduction (L64).

13b) Also your pipeline seems to use three different programming languages/environments... Any chance this could be reduced? Maybe there are R packages that can convert csv to matlab compatible formats, so you can avoid the Python step? (nothing against using the current pipeline per se, I am just thinking that for usability and adoption by other labs, the smaller amount of languages, the better?

This is a very important suggestion that highlights a clear limitation of the pipeline. I am happy to say that we worked on this and solved the problem integrating the Python version of Catch22 into the ethoscopy software. This means the two now integrate, and the entire analysis can be run within the Python ecosystem. HCTSA does not have a Python package unfortunately but we still streamlined the process so that one only has to go from Python to Matlab without passing through R. To be honest, Catch22 is the evolution of HCTSA and performs really well so I think that is what most users will want to use. We provide two supplementary notebooks to guide the reader through the process. One explains how to go from ethoscope data to an HCTSA compatible mat file. The other explains how ethoscope data integrate with Catch22 and provides many more examples than the ones found in the paper figures.

14) There are two sections named "References" (which are different from each other) on the manuscript I received and also on BioRxiv. Should one of them be a supplementary reference? Please correct it. I spent a bit of time trying to figure out why cited references in the paper had nothing to do with what was being described...

The second list of references actually applied only to the list of compounds in the supplementary table 1. When generating a collated PDF this appeared at the end of the document and created confusion. We have now amended the heading of that list in the following way, to read more appropriately:

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The authors have assembled an enormous amount of statistical data on the genomes and phylogeny of Arctic algae, including the genomes of four new species that they sequenced for this study. Their main finding is that horizontal gene transfer has led to convergent evolution in distantly related microalgae.

**Major comments**

Reviewer #1__: The purpose of the study is not clearly stated in the abstract or the introduction. The authors say (line 93) "Defining the genetic adaptations underpinning these small algal species is crucial as a baseline to understand their response to anthropogenic global change (Notz & Stroeve,2016)." Is this their goal? Or are they just quoting another study? The authors state (line 103) "We extend by sequencing the genomes of four distantly related microalgae...". This is not really a question or a hypothesis. I am sure the authors can provide a more compelling reason to embark on such a labor-intensive study.__

Reply: We agree that the aim was lost in the details and the Introduction is now focused towards the original goal of the study, which was to investigate convergent evolution in a biogeographically isolated ocean. Additional references on the formation and history of the Arctic Basin have been added to the Introduction to provide context. “An ocean has been present at the pole since the beginning of the Cretaceous. Shaped by tectonic processes (Nikishin et al., 2021) the Arctic Ocean has been a relatively closed basin since the Masstrichtian at the end of the late Cretaceous epoch (ca. 70 million years before present), with episodic sea-ice cover since that time (Niezgodzki et al., 2019). This long history suggests limited gene flow from the global ocean over vast time scales and Arctic marine species including microalgae could well have unique adaptations to cold arctic conditions.” Line 78-83.

And following this we provide a clear hypothesis “The potential for lineages of ancient Arctic origin and the episodic input of outside species led us to our hypothesis that Arctic microalgae convergently evolved traits or adaptations aiding survival in an ice-influenced ocean. Line 112-117.

We also discuss both the adaptive and distinct physical environment of the Arctic, and its topographical separation from other ocean regions as dispersal limitation would enhance the Arctic-specific genomic signatures. We now cite the recent paper by Sommeria-Kline et al. (2020), which puts eukaryotic plankton biogeography into a global context (Line 72)

Reveiwer #1__: The most prominent shared trait that the authors found are genes for ice-binding proteins. However, in view of their importance, little information is given about their different types and possible functions.__

Reply: We appreciate the comment and have added information on relevant ice binding proteins found in the Arctic Algae. In addition, we discuss how the functional and secretory diversity of IBP would enhance the survivability of pelagic taxa. Lines 534 to 564.

Although ice binding proteins from multicellular animals and plants are outside the scope of this study, there is a recent review; Bar Doley, Braslavsky and Davies 2016 Annual review of Biochemisty 85: 515-542.

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Reviewer #1__: The HGT of ice-binding proteins is a major focus of this study, but little is said about what previous studies have said about this. What are the previous studies, what are their findings and how do the present findings contribute to this?__

Reply: We agree that this aspect should have been more visible. We incorporated new data to characterize IBPs drawn from MMETSP transcriptomes, and environmental Tara Ocean metagenomes, as well as our Arctic strains. We note that as we take a PFAM-based approach, the IBPs treated are DUF3494/PF11999 domain, which are type 1 IBPs / algal IBPs (Raymond and Remia 2019). As an example of novelty, we identify the position of IBPs from dinoflagellates, within a larger Arctic Clade that included CCMP2293, CCMP2436 and CCMP2097 and Arctic TARA IBP, rendering this a pan-algal IBD clade.

In addition, we were able to resolve the position of anomalous F. cylindrus IBP that fell between two Arctic associated clades (A and B, in our Fig 4). This finding is consistent with F. cylindrus originating in the Arctic as previously suggested and subsequently invading the Southern Ocean.

The recurrent acquisition of multiple diverse IBP isoforms in individual species through HGT events has not been previously reported, and the extent of isoforms in the Arctic was surprising. See for example multiple different IBP forms with separate origins in Pavlovales CCMP2436 (Fig 4). The previous studies are referred to in the context of the phylogeny of the IBD within the results section: Lines 322- 413, and Lines 534-585.

Reviewer #1: Figure 5 on HGT of ice-binding proteins is difficult to follow. It would be clearer if each panel could be described separately, clearly stating its main finding. I doubt that a reader could look at this figure and explain to a colleague what it shows.

Reply: We have revised rearranged the figure (now Fig 4) with Arctic A, B, C and D clearly indicated as well as the two Antarctic dominated clades. The upper schematic includes the deepest phylogeny of algal IBDs to date, incorporating all of UniRef, MMETSP and TARA Oceans. The fasta files underlying the tree and the nexus file used are provided the S1 Data Folder, which is an excel folder with information on the analysis of the data. The callout and order of the clades has been revised to facilitate interpretation of the phylogenies more clearly. The entire section has been completely rewritten.

Reviewer #1: This is also a problem with many of the other figures. For each figure, what is the question being asked and what is its take-home message?

Reply: We agree that the message was lost and have now focused on our original question in our accepted proposal to JGI. “Is there a convergence among arctic microalgae at the genomic level?”. We found some genome properties were common among the Arctic isolates (more unknown PFAMS and several expanded PFAMs). The importance of ice binding proteins in Arctic Isolates and the widespread inter-algal HGT of this important protein among the Arctic strains. The IBP biogeography and phylogeny strongly indicate that the Arctic microalga have acquired IBP locally and that the Antarctic strains have acquired additional isoforms independently from Antarctic bacteria and fungi (Lines 565-585).

Reviewer ____#1____: ____The paper has more data than a reader can absorb. It could be strengthened by reducing the number of figures, simplifying them if possible, and more clearly stating the value of the remaining figures.

Reply. As suggested, we have refocused the paper, removing more speculative statistics based analysis and associated figures. The main conclusions are supported by the 5 main figures. We are now present 5 main figures and 11 supplementary figures (previously 23 downloadable supplementary figures and 40 on-line only figures supporting the support figures). We agree with the reviewer, and we feel the revised version is a more transparent synthesis. Briefly the Figures illustrate the following points. Fig. 1. The multigene tree of available algal genomes and transcriptomes provides a clear framework for judging the divergence of subsequent individual gene and PFAMs phylogenies. Fig. 2 (originally Fig. 3). Indicates the convergence of PFAM domains in the Arctic strains, in contrast to strains from elsewhere. Fig. 3 (originally Figure 4) shows Arctic specific expansions and contraction of PFAM domains, again demonstrating convergent evolution in the Arctic. The figure identifies specific PFAMs that contribute to the within-Arctic convergence. This figure is based on statistical methods independent of Fig 2. Figure 4 is the most extensive IBP phylogeny to date and has been discussed above. Figure 5, which was supplementary in our non-peer reviewed version, shows the biogeographic distribution of IBP, and can be compared to the distributions of the 18S rRNA genes from the four Arctic algae provided as supplementary (S6 Fig.)

**Minor comments**Reviewer #1

1. The figure citations are confusing. E.g., what does "Fig.1- Figure supplement 1" refer to? Does this refer to 1 or 2 figures? Apparently, it refers only to Fig. S1, so many readers will be confused when they look at Fig. 1.

Reply: We apologize for the confusing format; the manuscript had been formatted for the online journal eLife. Our revision follows the more traditional style of PLoS Biology and other Review Commons journals.

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Multiple citations should be in order of publication date, not alphabetical order.

Reply ; We agree that date of publications is quite standard and recognizes priority of publication. Several on line journals no longer follow this rule and citation order will follow the specific style used by our accepting journal.

Reviewer #1 (Significance (Required)): It is well known that useful genes tend to be shared among microorganisms. The present study strengthens previous studies in showing that gene transfer is an important process in polar regions.

Reply: We thank the reviewer for recognizing the importance of our study.

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Reviewer #2 ____(Evidence, reproducibility, and clarity (Required)):

This manuscript is the result of a large international collaborative effort, including the US Department of Energy Joint Genome Institute. Its focus is comparative genomics of eukaryotic Arctic algae. The primary data described in the ms are four new genome and transcriptome sequences from diverse Arctic algae, represented by a cryptomonad, a haptophyte, a chrysophyte, and a pelagophyte.

The authors compare these new data to previously published genomic/transcriptomic data from eukaryotic algae with the goal of understanding genome evolution in the Artic. The results of the paper are a series large-scale comparative genomic bioinformatics analyses, including the associated statistical analyses. The key findings center on statistically significant features of Arctic genomes, features that stand out as compared to the genomes of algae that are not primarily found in the Arctic. Together, these findings allow the authors to make various hypotheses and suggestions about genetic adaptations to polar environments.

By far the most significant finding is that the genomes of Arctic algae are enriched in genes encoding proteins with an ice-binding domain, paralleling findings from Antarctic algae. These genes appear to have spread among Arctic algal genomes via horizontal gene transfer, which raises a series of interesting questions. In my opinion, the major conclusions of this paper are supported by the data. Listed below are a few comments that may improve the ms:

Reviewer #2.

1) In today's post-genomics era, everyone seems to be sequencing nuclear genomes. Often what distinguishes high-impact and low-impact genome papers is the number of genomes presented and the quality of the genome assembly. I may have missed it, but reading the main text, the figures/tables, and the supplementary data I was not able to get a sense of the quality of the four genome assemblies from which the main findings are based. I was eventually able to find this information from PhycoCosm (note: some of the links to this site are not working in the ms). My quick scan of the PhycoCosm summary info for the four genomes indicates that the assemblies are highly fragmented, likely because they are based on short-read Illumina sequencing rather than a combination of short and long reads. I think it is important to briefly discuss (and or present) the quality of the assemblies in the ms and to highlight the potential limitations/drawbacks of employing highly fragmented assemblies when carrying out large-scale comparative genomics.

Reply: We agree and the data concerning the genome quality assemblies has been moved to the main text Table 1. The comparison with other paired related strains is provided in an excel folder designated S2 Data Folder.

Reviewer #2.

2) Horizontal gene transfer is undeniably a major driving force in evolution, and one that has shaped genomic architecture across the Tree of Life. I believe the data presented here support a role for HGT in the genome of evolution of Arctic algae, particularly with respect to genes encoding proteins with an ice-binding domain. However, we can all think of numerous instances when authors of genome papers were too quick to point to HGT. Thus, I would urge more caution and balance when presenting the HGT data, including some discussion about factors that could incorrectly lead researchers to conclude a significant role for HGT, such as contamination, gene duplication, mis-assemblies, etc. I'm not suggesting that you change the main conclusions, but just tone down the language in places (e.g., "we reveal remarkable convergence in the coding content ... ").

Reply: We understand the reviewers concerns and now more clearly outline the pipeline we have used to identify HGTs. This included: filtering each genome to remove all possible contaminant sequences first, considering both contig co-presence of vertical- and horizontally-derived genes, and reciprocal and independent annotations of gene sequences in both genome sequences and MMETSP transcriptomes. Retained genes were subjected to simultaneous BLAST analysis and manually curated phylogenies using decontaminated reference datasets. The most parsimonious explanation for our final IBP domain microbial algal clusters (Fig 4) is HGT. On the side of caution, we removed the entire section that identified potential arctic HGT based primarily on a less targeted broad statistical analysis. The focus is now on 3 genes that have clearly identifiable utility in the Arctic, were found to be enriched in Arctic genomes via a separate analysis and had homologs in the Tara Ocean Polar circle data. In addition, we describe more clearly the role of expansion and enrichment of PFAMs and the high proportion genes without an identifiable PFAMs in the Arctic strains as evidence for Arctic convergence separate from potential HGT.

Reviewer #2.

3) The downside of studying protists (as compared to multicellular animals, for instance) is that most are not widely known by the scientific community and even fewer scientists can picture what they actually look like (e.g., Pavlovales sp. CCMP2436). A few more details about the four Arctic algae that make up the focus of this paper might be helpful for the casual reader. My sense is that if at the next departmental meeting I asked my colleagues what a pelagophyte was most would look at me with a blank stare. Moreover, am I right to assume that all four algae are psychrotolerant rather than psychrophilic (Supplement Fig. 1 makes me think otherwise). It might be good to point out the difference in the text.

Reply: High resolution images of each strain are available on the JGI home page for each alga, given the multiple figures we feel photos would not add information.

Reviewer #2

4) I don't think Supp. Table 1 (the Pan-algal dataset) got uploaded correctly during the manuscript submission stage. The first link I click on gives me Supp. Table 2.

Reply: We apologize for this, the format was incorrect for the file designation and there were lost links. We now more actually refer to these as Data Folders as they are excel folders containing multiple sheets, All supplementary links will be verified again on final submission.

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Reviewer #2 (Significance (Required)):

By far the most significant finding from this paper is that the genomes of Arctic algae are enriched in genes encoding proteins with an ice-binding domain, paralleling findings from Antarctic algae. These genes appear to have spread among Arctic algal genomes via horizontal gene transfer, which raises a series of interesting questions. This is not the first paper to present these types of ideas, but it is arguably the broadest analysis yet, at least with respect to eukaryotic algae. This work will be of great interest to polar scientists, phycologists, protistologists, and the genomics community. I am genome scientist studying protists, including algae.

Reply. We thank the reviewer for their insightful comments.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

**Summary:**

This manuscript is focused on Arctic microalgae, an important yet understudied community in permanently cold ecosystems. By sequencing the genomes of four phylogenetically diverse and uncharacterized polar algae, the authors seek to elucidate genomic features and protein families that are similar in polar species (and differ from their relatives from temperate environments) This work used high-throughput genomic sequencing and computational analysis to demonstrate significant horizontal gene transfer (HGT) in several gene families, including ice-binding proteins. The authors suggest that this HGT is an effector of environmental adaptation to Arctic environments.

**Major comments and experiment suggestions:**

The authors conclude that HGT between arctic species is a driver of polar adaptation. The authors strongly support the claim that HGT is present more frequently in the polar algae examined here. Whether this is adaptive should be further explored though. For instance, ice-binding domains were one PFAM group found at significantly higher frequencies in the polar species - but are all of these species associated with ice? What would be the benefit of IBDs in an alga that is found in the open ocean. Similar with the other domains (Lns 333-335), its not clear whether these are truly adaptive features. ____This is more speculative.

Reply: We agree that detail was lacking and have considerably expanded our introduction on the character of the Arctic Ocean and have stated the goals and underlying hypothesis. Briefly, all surface water organisms that live in the Arctic encounter ice during the year as the ocean freezes in winter, and surface waters reman around negative 1.7 °C for much of the year. This information has been added to the introduction. We have also expanded the discussion on the multiple effects of different IBPs that would be ecologically beneficial for plankton as well as ice-algae and cite relevant experimental studies and reviews.

Reviewer #3) ____HGT was a major conclusion of this study, putting this in a wider perspective would strengthen the conclusion, especially in the context of HGT from prokaryotes. Are there insights on whether IBDs are present in Arctic prokaryotes?

Reply: This is a good question, and we now point out that there were 91 Arctic bacterial and archaeal IBP sequences in our comparative dataset. In contrast to the Antarctic clades, none were closely related to the Arctic strain IBPs (Fig 4). Line 336.

Reviewer #3) ____The data obtained from the genomic works supports the conclusions stronger that ones from transcriptomes, where what genes/domains are present would depend largely on the sampling conditions. This should be emphasized.

Reply: The main rational for using transcriptomes was that more of these are available and enabled us to detect convergences and HGT across a broader taxonomic range than would be possible with genome-only data, where we had access to a total of only 21 microalgal genomes. In general transcriptome studies are aimed at identifying responses under different conditions and rely on comparative expression data, usually 2-fold differences in up or down expression under different growth conditions, see for example Freyria et al. 2022 (Communications Biology). Unlike a transcriptome expression study, our data mining detected any (constitutive or regulated) expression in these unicellular haploid cells, we would have detected genes used under any condition that an algal happened to be growing. IBD was not detected in any of the temperate genomes, and only detected in transcriptomes of Arctic and Arctic-Boreal groups. However, we agree that there may be some limitation of transcriptomes only studies and mention this. Lines 522-528.

Reviewer #3) ____An experiment to determine whether the species are cold extremophiles (psychrophiles) would be useful here to strongly support the data in Figure 1. The authors state that their species can not survive >6C but this is based on experiments done on older studies. Considering the cultures have been maintained as a continuous culture for decades, confirming that they still have psychrophilic characteristic would be useful. This is a straightforward and low cost experiment that requires simply measuring growth rates at several temperatures to define the optimal and confirm that the cells are not viable above 6C.

Reply: These are interesting points, and the broad “background” statements in the original manuscript would require a separate study,and have been deleted. Temperature tolerance experiments are not so simple for cold adapted algae with slow growth rates. Such experiments require specialized incubators to maintain low temperatures. Temperature experiments have been carried out on the cultures in the context of other studies, see for example, Daugberg et al. 2018, J. Phycol. But this is not within the scope of the present study.

We now restrict our conclusions to the specific question of convergence among Arctic strains. We apologize for the misunderstanding on the history of the cultures. They have not been in “continuous culture” but are cryopreserved. We now simply indicate that they grow below 6 °C, which is sufficient to assume that they are likely cryophiles, our experience is that they do not grow well or at all at higher temperatures, our efforts have been to maintain the cultures that are otherwise easily lost. We now make no claims about optimality or limits. Here we simply examined genomes and available transcriptomes that were generated from algae growing at 4-6 °C.

Reviewer #3) ____**Minor comments:**

Defining the species used here as psychrophiles would put the study in context better. The authors relate their finding to Antarctic species (HGT, ice-binding domains, large genomes) all of which are confirmed psychrophiles.

Reply: The temperature definition of psychrophiles is surprisingly high (optimal growth below 15 °C) and this definition of psychrophiles is now given in the introduction. The point is really that there are few isolates from cold surface waters that have been well studied. We now add. “A handful of polar algal genomes have been extensively studied, with 4 of these from around Antarctica and classified as psychrophiles (not being able to grow above 15 °C (Feller & Gerday, 2003)”. Lines 103-107.

Reviewer #3) ____A short rationale on why these species at all would be useful - are they representative of their classes? Do they have psychrophilic characteristics that might make them useful models in the future? Are they widely used now?

Reply: We appreciate the point as the definition of utility in discovery-based science is an open dialog.

We agree that the study requires context and have added our rational for selecting the species for genome sequencing to the introduction. “To address questions on genetic adaptations to this ice-influenced environment, we sequenced 4 phylogenetically divergent microalgae, from 4 algal classes belonging to 3 algal phyla: Cryptophyceae (Cryptophyta), Pavlovophyceae (Haptophyta), Chrysophyceae and Pelagophyceae (both in the Ochrophyta) isolated from the ca. 77 °N, where surface ice flow persists through June (Mei et al., 2002). The four isolates were selected as representatives of different water and ice conditions and phylogeny from available strains collected in April and June 1998 during the North Water Polynya study”.

Reviewer #3) ____Starting algal cultures were maintained in a continuous culture since 1998 and under continuous light since at least 2015, have the authors confirmed that these algae retain their physiological features even after this long time? The accumulation of mutations is a possibility here.

Reply: We apologize for the misunderstanding of the timeline; the history of the cultures was not given in the manuscript and the inferred history is not quite correct. The 2015 date was the year of publication for the MMETSP data. Our continuous light statement is a record of our standard culture conditions. We now elaborate on the material used in the current study. The cultures were deposited in the Bigelow culture collection (now NCMA) in 2002 and cryopreserved once they had been verified and given a culture designation. We obtained fresh cultures in 2005 and these were used for the MMETSP project. We obtained fresh cultures again in 2011, specifically for the JGI genome project. These algae do not grow fast and most of the DNA was sent to JGI in 2012 for most of the isolates. This history is rather long and not relevant, since one would speculate that over the years the algae would tend to lose the ice associated functionality, e.g. they were not frozen in seawater every year for 4 to 6 months or subject to sudden freshwater exposure, when ice melts. We would encourage other researchers to order the cultures and run experiments. We note that many of the 40 or so algae isolated from the same campaign have been used by others for specific studies and at least 8 are in the MMETSP data set. The presence of 18S rRNA and phylogenetic position of the IBP sequences compared to Tara Arctic circle data confirms long-term Arctic presence of each species and the IBP domains in the Arctic without marked changes over the last 20 years.

Reviewer #3) ____Ln381 - The culture collection IDs for each sequenced species should be included here

Reply: we have added the culture IDs throughout.

Reviewer #3) ____Ln. 389 - Algal cells are harvested and used for nucleic acid extraction, the nucleic acids themselves are not harvested

Reply: we agree and corrected the wording

Reviewer #3 (Significance (Required)):

This study is well places in the current state of research on polar alga and represents a significant and very valuable addition to the current knowledge pool. Algae in general are lagging behind other groups of photosynthetic organisms in the number of sequenced and analyzed genomes, despite algae being one of the main primary producers globally. This is even more strongly felt in polar research, where only 4 species have been sequenced, most of which are restricted to Antarctica. There is a true gap in our knowledge when it comes to Arctic species, and this study fills this gap. As the authors correctly state, we need more knowledge on polar environments and the primary producers that support these important ecosystems in light of current climate change trends.

Reply: we appreciate the succinct summary of our study and thank the reviewer for insights and suggestions that have improved the manuscript.

Reviewer field of expertise: Polar algae, stress responses, plant and algal energetics, cell signalling

Reply: We appreciate the incites and perspective steming from the reviewer's expertise.

Relevant key references cited in the reply:

Daugbjerg N, Norlin A, Lovejoy C. Baffinella frigidus gen. et sp. nov. (Baffinellaceae fam. nov., Cryptophyceae) from Baffin Bay: Morphology, pigment profile, phylogeny, and growth rate response to three abiotic factors. Journal of Phycology. 2018;54(5):665-80

Feller, G. and Gerday, C. (2003) Psychrophilic enzymes: Hot topics in cold adaptation. Nat Rev Microbiol, 1, 200-208.

Freyria NJ, Kuo A, Chovatia M, Johnson J, Lipzen A, Barry KW, et al. Salinity tolerance mechanisms of an Arctic Pelagophyte using comparative transcriptomic and gene expression analysis. Communications Biology. 2022;5(1). doi: 10.1038/s42003-022-03461-2

Mei, Z. P., Legendre, L., Gratton, Y., Tremblay, J. E., Leblanc, B., Mundy, C. J., Klein, B., Gosselin, M., Larouche, P., Papakyriakou, T. N., Lovejoy, C. and Von Quillfeldt, C. H. (2002) Physical control of spring-summer phytoplankton dynamics in the North Water, April-July 1998. Deep-Sea Research Part Ii-Topical Studies in Oceanography, 49, 4959-4982.

Niezgodzki, I., Tyszka, J., Knorr, G. and Lohmann, G. (2019) Was the Arctic Ocean ice free during the latest Cretaceous? The role of CO2 and gateway configurations. Global and Planetary Change, 177, 201-212.

Nikishin, A. M., Petrov, E. I., Cloetingh, S., Freiman, S. I., Malyshev, N. A., Morozov, A. F., Posamentier, H. W., Verzhbitsky, V. E., Zhukov, N. N. and Startseva, K. (2021) Arctic Ocean Mega Project: Paper 3-Mesozoic to Cenozoic geological evolution. Earth-Science Reviews, 217.

https://tinysubversions.com/spring-83/rrffcc-dfk-1.txt

Some interesting points from someone with experience on several fronts.

I love that he's published his response in plain text this way!!

I thought of the same aesthetic as well, in part because it wasn't as "busy" as the comic book page aesthetic.

https://www.zylstra.org/blog/2022/06/spring-83/

I've been thinking about this sort of thing off and on myself.

I too almost immediately thought of Fraidyc.at and its nudge at shifting the importance of content based on time and recency. I'd love to have a social reader with additional affordances for both this time shifting and Ton's idea of reading based on social distance.

I'm struck by the seemingly related idea of @peterhagen's LindyLearn platform and annotations: https://annotations.lindylearn.io/new/ which focuses on taking some of the longer term interesting ideas as the basis for browsing and chewing on. Though even here, one needs some of the odd, the cutting edge, and the avant garde in their balanced internet diet. Would Spring '83 provide some of this?

I'm also struck by some similarities this has with the idea of Derek Siver's /now page movement. I see some updating regularly while others have let it slip by the wayside. Still the "board" of users exists, though one must click through a sea of mostly smiling and welcoming faces to get to it the individual pieces of content. (The smiling faces are more inviting and personal than the cacophony of yelling and chaos I see in models for Spring '83.) This reminds me of Stanley Meyers' frequent assertion that he attempted to design a certain "sense of quiet" into the early television show Dragnet to balance the seeming loudness of the everyday as well as the noise of other contemporaneous television programming.

The form reminds me a bit of the signature pages of one's high school year book. But here, instead of the goal being timeless scribbles, one has the opportunity to change the message over time. Does the potential commercialization of the form (you know it will happen in a VC world crazed with surveillance capitalism) follow the same trajectory of the old college paper facebook? Next up, Yearbook.com!

Beyond the thing as a standard, I wondered what the actual form of Spring '83 adds to a broader conversation? What does it add to the diversity of voices that we don't already see in other spaces. How might it be abused? Would people come back to it regularly? What might be its emergent properties?

It definitely seems quirky and fun in and old school web sort of way, but it also stresses me out looking at the zany busyness of some of the examples of magazine stands. The general form reminds me of the bargain bins at book stores which have the promise of finding valuable hidden gems and at an excellent price, but often the ideas and quality of what I find usually isn't worth the discounted price and the return on investment is rarely worth the effort. How might this get beyond these forms?

It also brings up the idea of what other online forms we may have had with this same sort of raw experimentation? How might the internet have looked if there had been a bigger rise of the wiki before that of the blog? What would the world be like if Webmention had existed before social media rose to prominence? Did we somehow miss some interesting digital animals because the web rose so quickly to prominence without more early experimentation before its "Cambrian explosion"?

I've been thinking about distilled note taking forms recently and what a network of atomic ideas on index cards look like and what emerges from them. What if the standard were digital index cards that linked and cross linked to each other, particularly in a world without adherence to time based orders and streams? What does a new story look like if I can pull out a card either at random or based on a single topic and only see it or perhaps some short linked chain of ideas (mine or others) which come along with it? Does the choice of a random "Markov monkey" change my thinking or perspective? What comes out of this jar of Pandora? Is it just a new form of cadavre exquis?

This standard has been out for a bit and presumably folks are experimenting with it. What do the early results look like? How are they using it? Do they like it? Does it need more scale? What do small changes make to the overall form?

---

For more on these related ideas, see: https://hypothes.is/search?q=tag%3A%22spring+%2783%22

Pretty basic and pretty much what Jaiku did. It gave a somewhat holistic social platform view of people with the person in focus. You could see all the social platforms they shared on and what was shared, but also allowed you to turn off listening to platforms for that person.

On the Jaiku webview I had a my own layer of how I had someone tagged (say foodie or Apple nerd) and I could click on that and seem others I followed who I believed had those interests. Which was a personal version of what I wrote up later as a Granular Social Network (https://www.vanderwal.net/random/entrysel.php?blog=1975).

the limit on history is interesting and feels like a missed opportunity. in a world where storage is so cheap, we should be able to keep our own histories! thinking about sousveillance vs. surveillance and personal panopticons

reminds me of locket but web-based and with full user agency to create what they want. their own little pocket of the internet. Love the no account part and think that's super important..

https://www.robinsloan.com/lab/specifying-spring-83/

This is essentially the SSB take too. I don't love it, but the way in which I don't love it is a crotchety computer professional way, not a meaningful one.

Peer to peer content transmission is an awfully big can of worms. Freedom to delete is real contentious in SSB/Fediverse sstuff.

One uncomfortable thing about this is that it replicates a scarcity logic of space in an unscarce medium. I keep lots of barely-more-than-defunct feeds in my RSS reader because it costs me nothing to do so and I'd be so happy if they did start publishing again. Looking at an unchanged board for a year would feel different.

This is fun! Maybe I should try sketching some stuff out here before I work further on that web directory SSG template project.

Reminds me of the neocities webgardens (that list of creators is incomplete), doing something like this with iframes and friendly convention.

But -- external images? No images? 😔

looks around shiftily at RSS reader

Viva! I do still use RSS as more of a notification stream than a consumption stream; I don't really want the consumption to all happen in one place. When Cinni updates in her RSS feed, it's links to her proper pages, and this is right and good because her site is beautiful in a way RSS couldn't (shouldn't) accommodate.

Hmm. I don't know that there's a way to get around the 90-9-1 ratios there. I had to resort to user scripts even for my own stuff. I'm reminded of this whole thing, too.

I am forcibly reminded of this orange site comment that makeworld had shared though I guess I haven't really gotten it out of my head in the first place.

why that number?

This is probably where Fraidycat falls down. But still, as you don't mention it, Robin, are you familiar with it?

Just follow? Not interact with? Build relationships with?

First thought: https://fraidyc.at/

But I expect the discussion below to clarify how that does not suffice (?).

Literally laughed out loud to this.

This seems like a good opportunity to try hypothes.is again. I've been meaning to get back to using it. As an experiment, I'm going to try and annotate this post as I read it.

https://www.jeremycherfas.net/blog/spring-it-on

https://www.louispotok.com/spring83

https://tracydurnell.com/2022/06/10/following-people-outside-feed/

is it? I feel like you could certainly participate with raw markdown styled with Robin's defaults. hard to know how people who aren't Like Us would use the form because of course it's people Like Us who want to

I wonder if, given that boards are supposed to expire anyway, the key rotation shouldn't involve publishing old secrets such that it's not possible to use an old key to pin someone down as having published something (which would be long after its intended expiry anyway)? With the caveat that I don't know nothing about cryptography, I'm just a horrible little goblin typing in a Hypothes.is pane.

This is interesting. I could imagine wanting a more instant version of a tiled view of those I follow -- back when I was on Twitter, there was something cool about the implicit group liveblog of e.g. a major news event. I would have liked to be able to see it all tiled out in one view.

I'm not sure implementation difficulty is the same thing as simplicity. Gemini also says those things are the same. This is also really tricky to evaluate in a world of libraries and linking; we're not mentally including the difficulty of Ed25519 cryptography in our assessment of the protocol's "simplicity" because we're not absolute loons and would use a library for that... but it's part of the fabric being woven.

My gut desire is always for social media accommodating closer to Darius's 50-ish, Dunbar's tiers. I wonder how allowing for media publishers needs to change those numbers.

Is this really compatible with "[embracing] the richness, flexibility, and chaos of modern HTML and CSS"? I don't have a big negative opinion about it because fixed display size is how webgardens work, too, but articulating the root of the design decision (to allow things to tile nicely?) could open interesting discussion.

sobs

This has chafed at the users of Gemini even while its designers seem to be proud of holding the line.

Yeah, ditto.

Author Response:

Reviewer #1 (Public Review):

In this manuscript by Gilbert et al., the authors found that MLC1 is required for postnatal maturation of perivascular astrocyte coverage. Through various detailed experiments, they further found that Mlc1 KO mice showed a number of defects, including the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow. The data is well presented, however there are several points that need to be addressed to strengthen the manuscript.

1) Since many PvAP proteins showed the normal expression after P60 in Mlc1 KO mice, it is also possible that many of the phenotype that the authors presented, such as the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow, can be recovered after P60. This would be important as well to understand the prime pathological cause of megalencephalic leukoencephalopathy induced by MLC1 deletion.

Although some protein levels are normal in Mlc1 KO mice, PvAP coverage and gliovascular unit morphology are altered at P60. We also now show (using TEM) that PvAPs are swollen in 1-year-old Mlc1 KO mice (the new Fig. S4) - indicating that edema develops progressively in Mlc1 KO mice. Myelin vacuolation starts at 3 months and progressively worsens (Dubey et al., 2015). Taken as a whole, these data show that MLC is a degenerative disease. Under these conditions, the recovery of gliovascular unit function after P60 is very unlikely.

All the molecular morphological and functional changes in gliovascular unit described in our manuscript precede myelin degradation, which starts at the age of 3 months in Mlc1 KO mice (Dubey et al., 2015). Moreover, our previous study (Gilbert et al., 2019) demonstrated that MLC1 expression starts around P5 and that the MLC1/GlialCAM complex is only mature at P15. Taken together, these results strongly suggest that gliovascular unit alterations are the primary pathological events in MLC.

Dubey M, Bugiani M, Ridder MC, Postma NL, Brouwers E, Polder E, Jacobs JG, Baayen JC, Klooster J, Kamermans M, et al. 2015. Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle. Ann Neurol 77: 114-131.10.1002/ana.24307. Gilbert A, Vidal XE, Estevez R, Cohen-Salmon M, and Boulay AC. 2019. Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation. Brain Struct Funct 224: 1267-1278.10.1007/s00429-019-01832-w.

2) Does CBF get differed only after neuronal stimulation in Mlc1 KO mice? It is unclear whether the basal CBF/neurovascular coupling level is disrupted as well in Mlc KO brains and how this defect is related to the reduced vasoconstriction in these mice.

The baselines of the functional ultrasound experiments were aligned prior to stimulation. This technique measures the percentage increase in blood flow after neuronal stimulation (here, whisker movement) but does not measure the basal flow and does not enable one to distinguish between an abnormal basal cerebral blood flow (as suggested by the reduction of the arterial diameter) and the loss of vascular contractility - both of which probably contribute to the defect in neurovascular coupling.

3) The reduced cohesiveness of PvAPs and the associated neuronal fibers to the vessel in Mlc1 KO brains should be validated with additional experimental approach.

To strengthen our analysis, we now give the results of a parallel quantitative immunofluorescence analysis of purified brain vessels (presented in a new figure, Fig.5). The results show that part of the Aqp4 and NF-M perivascular immunolabeling is absent in the Mlc1 KO. Taken as a whole, our data demonstrate that PvAPs and the associated neuronal fibers (which normally remain attached to brain vessels during mechanical purification) are lost during the purification process in Mlc1 KO mice but not in the WT. In conclusion, the absence of MLC1 reduces the mechanical cohesiveness of PvAPs and the associated neuronal fibers.

4) The defective polarity of astrocytes should be better described by using other markers other than GFAP. The distribution of Aquaporin4, Cx43 or several glutamate transporters in the specific compartment of astrocytes can be examined.

GFAP is the marker typically used to analyze the astrocytes’ overall morphology and polarity. Nevertheless, we agree that it is of interest to study the molecular polarity of PvAPs. Indeed, morphological changes in the PvAPs and astrocytes and changes in polarity in Mlc1 KO might all influence the localization of molecules in PvAPs. To address this question, we performed a quantitative stimulated emission depletion (STED) analysis of protein localization in PvAPs. Our results indicate that the perivascular localization of aquaporin 4 was not affected. However, the density and size of Cx43 puncta were greater - indicating that the gap junctions in PvAPs are not organized in the same way in the Mlc1 KO as in the WT. This observation is consistent with our electron microscopy observations of perivascular astrocytic processes stacked on the top of each other and linked by extended gap junctions.

We have also added results for Kir4.1, a potassium channel that is expressed preferentially in PvAPs. The Kir4.1 expression level in Mlc1 KO was lower at all stages of development, indicating that perivascular potassium homeostasis was probably perturbed. These results are interesting because (i) epilepsy is a significant component of megalencephalic leukoencephalopathy (Dubey et al., 2018; Yalcinkaya et al., 2003), and (ii) Kir4.1 deletion or downregulation is associated with greater susceptibility to epilepsy (Sibille et al., 2014). These points are now discussed.

Dubey M, Brouwers E, Hamilton EMC, Stiedl O, Bugiani M, Koch H, Kole MHP, Boschert U, Wykes RC, Mansvelder HD, et al. 2018. Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts. Ann Neurol 83: 636- 649.10.1002/ana.25190. Sibille J, Pannasch U, and Rouach N. 2014. Astroglial potassium clearance contributes to short-term plasticity of synaptically evoked currents at the tripartite synapse. J Physiol 592: 87- 102.jphysiol.2013.261735 [pii] 10.1113/jphysiol.2013.261735. Yalcinkaya C, Yuksel A, Comu S, Kilic G, Cokar O, and Dervent A. 2003. Epilepsy in vacuolating megalencephalic leukoencephalopathy with subcortical cysts. Seizure 12: 388-396.10.1016/s1059-1311(02)00350-3.

5) The authors provide interesting observations such that the formation of perivascular astrocyte coverages is required for the dissociation of the contacts between neuronal components and the vessel during development. The authors need to discuss more about potential regulation and implication of this phenomenon.

This is indeed a fascinating phenomenon. The postnatal period is also an intense synaptogenic phase in the mouse brain (Chung et al., 2015), during which astrocytes and neurons might compete for the perivascular space. In the absence of MLC1 and thus PvAPs, the neurons might expand into the free space. We now comment on this point.

Chung WS, Allen NJ, and Eroglu C. 2015. Astrocytes Control Synapse Formation, Function, and Elimination. Cold Spring Harb Perspect Biol 7: a020370.cshperspect.a020370 [pii] 10.1101/cshperspect.a020370.

6) It is interesting that DOTA-Gd tracer shows different traces in Mlc1 KO brains. However, it is unclear how MLC1 deletion affects glymphatic system. Does the tracer normally enter to the perivascular spaces in Mlc KO brains? Does the tracer leak out more from the perivascular spaces in Mlc1 KO mice? Is the general clearance or drainages of the tracer impaired in Mlc1 KO mice? Would these defects be originated by the reduced perivascular astrocyte coverage or the reduced vasoconstriction itself?

Paravascular transport (as revealed by the injection of a tracer into the CSF) depends mainly on dispersion of the tracer in the subarachnoid space (SAS), the cisternae, and the parenchyma (including the interstitial and perivascular spaces). The uneven, slow dispersion of the tracer within the SAS (compared with dispersion in the blood) means that the tracer’s kinetics in the parenchyma are regiondependent. These differences can be accentuated by regional differences in the anatomy of the brain’s vasculature, i.e. the presence or absence of a perivascular space and the vessel’s topology. Lastly, the amount of DOTA-Gd available for diffusion within the parenchyma depends directly on its local concentration in the SAS. This can be seen on our contrast concentration maps (see Fig. 8), where the highest DOTA-Gd concentrations are found near the injection site (the cisterna magna), in line with previous reports (Iliff et al., 2012). In the Mlc1 KO model, dispersion of DOTA-Gd is presumably affected in the SAS and the parenchyma.

With regard to tracer dispersion in the SAS and the cisternae, our anatomical MRI showed that the brain volume is greater in the Mlc1 KO mouse than in the WT (see Fig. 1). These variations in the geometry of the SAS may account for much of the difference between the Mlc1 KO mice and WT mice. Although tracer concentrations appear to be similar in the cerebellum (close to the injection site), they are much lower in the more distant septal area of Mlc1 KO mice - suggesting that tracer transport within the SAS is restricted.

With regard to parenchymal dispersion, we showed that MLC1 is essential for the position of the astrocytes’ perivascular endfeet. Thus, in Mlc1 KO mice, the formation of the perivascular space (as a conduit for solute distribution) is likely to be deficient. This aspect is revealed by the slope of the tracer’s concentration-time curve, which indicate slower kinetics in Mlc1 KO mice; this might be due to poor integrity of the perivascular space. The higher volume of fluid in the Mlc1 KO parenchyma (reflected by the increased apparent diffusion coefficient (ADC); Fig. 1 and S1) might also be involved in this phenotype.

The heart beat is the main driver of CSF circulation in the perivascular space (Iliff et al., 2013). The heart rate is very rapid and so the heart exerts a much greater driving force on the CSF than the vasodilation of the vessels induced by neuronal activity. Alterations in vascular contractility observed in Mlc1 KO mice might be involved in the impaired CSF flux but this is unlikely.

All these points are now discussed in the revised version of the manuscript.

Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, and Benveniste H. 2013. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. Journal of Clinical Investigation 123: 1299-1309.10.1172/jci67677. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, et al. 2012. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med 4:147ra111.10.1126/scitranslmed.3003748.

Reviewer #2 (Public Review):

This very interesting manuscript by Gilbert and colleagues uncovers that the astrocyte specific membrane protein MLC1, the mutation of which causes a rare disease called megalencephalic leukoencephalopathy with subcortical Cysts (MLC), plays a fundamental role in the postnatal development of the gliovascular unit and the organization of the perivascular astrocyte processes, in particular. To reach this conclusion, the authors used an elegant multiscale approach including in vivo MRI, in vivo functional ultrasound, ex vivo analysis of vascular constriction, anatomical approaches at the light and electron microscopic level, and molecular characterization of the gliovascular unit from isolated microvessels. The manuscript is very well-written although it uses too many (unnecessary) abbreviations, which prevents a fluid reading of the manuscript, results are well illustrated and convincing and the discussion is reasonable.

I have a major concern regarding the results reported in Figure 4D, which seem somewhat contradictory to those shown in Figure 6A-F. Indeed, the authors report in Figure 4D that there is less Neurofilament-M protein around isolated microvessels in MLC1 KO mice, whereas Figure 6A-F shows that these animals have more neuronal processes in contact with the vessels than in wiltypes. How can the authors explain this?

The two situations are not comparable. On one hand, we observed the structure of the gliovascular unit in situ in fixed tissues. On the other, we mechanically purified microvessels. The detachment of astrocytic processes and associated neuronal fibers (linked to the mechanical dissociation of microvessels in the Mlc1 KO mouse) was clearly not counterbalanced by the presence of neuronal fibers contacting the vessels.

6.2.15. WIRE ROPE When power source and load are located at extreme distances from one another, or loads are very large, the use of wire rope is suggested. Design and use decisions pertaining to wire ropes rest with the user, but manufacturers generally will help users toward appropriate choices. The following material, based on the Committee of Wire Rope Producers, "Wire Rope User's Manual," current edition, may be used as an initial guide in selecting a rope.

Wire rope is composed of (1) wires to form a strand, (2) strands wound helically around a core, and (3) a core. Classification of wire ropes is made by giving the number of strands, number of minor strands in a major strand (if any), and nominal number of wires per strand. For example 6 × 7 rope means 6 strands with a nominal 7 wires per strand (in this case no minor strands, hence no middle number). A nominal value simply represents a range. A nominal value of 7 can mean anywhere from 3 to 14, of which no more than 9 are outside wires. A full rope description will also include length, size (diameter), whether wire is preformed or not prior to winding, direction of lay (right or left, indicating the direction in which strands are laid around the core), grade of rope (which reflects wire strength), and core. The most widely used classifications are: 6 × 7, 6 × 19, 6 × 37, 6 × 61, 6 × 91, 6 × 127, 8 × 19, 18 × 7, 19 × 7. Some special constructions are: 3 × 7 (guardrail rope); 3 × 19 (slusher); 6 × 12 (running rope); 6 × 24 and 6 × 30 (hawsers); 6 × 42 and 6 × 6 × 7 (tiller rope); 6 × 3 × 19 (spring lay); 5 × 19 and 6 × 19 (marlin clad); 6 × 25B, 6 × 27H, and 6 × 30G (flattened strand). The diameter of a rope is the circle which just contains the rope. The right-regular lay (in which the wire is twisted in one direction to form the strands and the strands are twisted in the opposite direction to form the rope) is most common. Regular-lay ropes do not kink or untwist and handle easily. Lang-lay ropes (in which wires and strands are twisted in the same direction) are more resistant to abrasive wear and fatigue failure.

Cross sections of some commonly used wire rope are shown in Fig. 6.2.123. Figure 6.2.124 shows rotation-resistant ropes, and Fig. 6.2.125 shows some special-purpose constructions.

Figure 6.2.123 Cross sections of some commonly used wire rope construction. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/d03c7ddb475211da/158e7be668be00fdf011bdadd2ef3b2300f542e92687c6e02a998fc06e5a48d6/06x02_123.png Open in new tab Share Figure 6.2.124 Cross section of some rotation-resistant wire ropes. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/e19ef67be879c4d0/9ab225782b323010f79e566c12a458512274145d6877d8209774da72c34eda94/06x02_124.png Open in new tab Share Figure 6.2.125 Some special constructions. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/8627b14662f8a537/bb284940d47ada0df68e2027b63f42e8b5f813ef1a6eb57a1dc9e1bdcb726b5b/06x02_125.png Open in new tab Share The core provides support for the strands under normal bending and loading. Core materials include fibers (hard vegetable or synthetic) or steel (either a strand or an independent wire rope). Most common core designations are: fiber core (FC), independent wire-rope core (IWRC), and wire-strand core (WSC). Lubricated fiber cores can provide lubrication to the wire, but add no real strength and cannot be used in high temperature environments. Wire-strand or wire-rope cores add from 7 to 10 percent to strength, but under nonstationary usage tend to wear from interface friction with the outside strands. Great flexibility can be achieved when wire rope is used as strands. Such construction is very pliable and friction resistant. Some manufacturers will provide plastic coatings (nylon, Teflon, vinyl, etc.) upon request. Such coatings help provide resistance to abrasion, corrosion, and loss of lubricant. Crushing refers to rope damage caused by excessive pressures against drum or sheave, improper groove size, and multiple layers on drum or sheave. Consult wire rope manufacturers in doubtful situations.

Wire-rope materials and their strengths are reflected as grades. These are: traction steel (TS), mild plow steel (MPS), plow steel (PS), improved plow steel (IPS), and extra improved plow (EIP). The plow steel strength curve forms the basis for calculating the strength of all steel rope wires. American manufacturers use color coding on their ropes to identify particular grades.

The grades most commonly available and tabulated are IPS and EIP. Two specialized categories, where selection requires extraordinary attention, are elevator and rotation-resistant ropes.

Elevator rope can be obtained in four principal grades: iron, traction steel, high-strength steel, and extra-high-strength steel.

Bronze rope has limited use; iron rope is used mostly for older existing equipment.

6.2.15.1. Selection of Wire Rope Appraisal of the following is the key to choosing the rope best suited to the job: resistance to breaking, resistance to bending fatigue, resistance to vibrational fatigue, resistance to abrasion, resistance to crushing, and reserve strength. Along with these must be an appropriate choice of safety factor, which in turn requires careful consideration of all loads, acceleration-deceleration, shocks, rope speed, rope attachments, sheave arrangements as well as their number and size, corrosive and/or abrasive environment, length of rope, etc. An approximate selection formula can be written as:

DSL

(NS) K b K sf where DSL (demanded static load) = known or dead load plus additional loads caused by sudden starts or stops, shocks, bearing friction, etc., tons; NS (nominal strength) = published test strengths, tons (see Table 6.2.65); Kb = a factor to account for the reduction in nominal strength due to bending when a rope passes over a curved surface such as a stationary sheave or pin (see Fig. 6.2.126); Ksf = safety factor. (For average operation use Ksf = 5. If there is danger to human life or other critical situations, use 8 ≤ Ksf ≤ 12. For instance, for elevators moving at 50 ft/min, Ksf = 8, while for those moving at 1,500 ft/min, Ksf = 12.)

Table 6.2.65 Selected Values of Nominal Strengths of Wire Rope Classification

Nominal diameter

Fiber core

IWRC

Approximate mass

Nominal strength IPS

Approximate mass

Nominal strength

IPS

EIP

in

mm

lb/ft

kg/m

tons

t

lb/ft

kg/m

tons

t

tons

t

Source: "Wire Rope User's Manual," AISI, adapted by permission.

6 × 7 Bright (uncoated)

¼

6.4

0.09

0.14

2.64

2.4

0.10

0.15

2.84

2.58

3 / 8

9.5

0.21

0.31

5.86

5.32

0.23

0.34

6.30

5.72

½

13

0.38

0.57

10.3

9.35

0.42

0.63

11.1

10.1

⅝

16

0.59

0.88

15.9

14.4

0.65

0.97

17.1

15.5

⅞

22

1.15

1.71

30.7

27.9

1.27

1.89

33.0

29.9

1⅛

29

1.90

2.83

49.8

45.2

2.09

3.11

53.5

48.5

1 3 / 8

35

2.82

4.23

73.1

66.3

3.12

4.64

78.6

71.3

6 × 19 Bright (uncoated)

¼

6.4

0.11

0.16

2.74

2.49

0.12

0.17

2.94

2.67

3.40

3.08

3 / 8

9.5

0.24

0.35

6.10

5.53

0.26

0.39

6.56

5.95

7.55

6.85

½

13

0.42

0.63

10.7

9.71

0.46

0.68

11.5

10.4

13.3

12.1

⅝

16

0.66

0.98

16.7

15.1

0.72

1.07

17.7

16.2

20.6

18.7

⅞

22

1.29

1.92

32.2

29.2

1.42

2.11

34.6

31.4

39.8

36.1

1⅛

29

2.13

3.17

52.6

47.7

2.34

3.48

56.5

51.3

65.0

59.0

1 3 / 8

35

3.18

4.73

77.7

70.5

3.5

5.21

83.5

75.7

96.0

87.1

1⅝

42

4.44

6.61

107

97.1

4.88

7.26

115

104

132

120

1⅞

48

5.91

8.8

141

128

6.5

9.67

152

138

174

158

2⅛

54

7.59

11.3

179

162

8.35

12.4

192

174

221

200

2 3 / 8

60

9.48

14.1

222

201

10.4

15.5

239

217

274

249

2⅝

67

11.6

17.3

268

243

12.8

19.0

288

261

331

300

6 × 37 Bright (uncoated)

¼

6.4

0.11

0.16

2.74

2.49

0.12

0.17

2.94

2.67

3.4

3.08

3 / 8

9.5

0.24

0.35

6.10

5.53

0.26

0.39

6.56

5.95

7.55

6.85

½

13

0.42

0.63

10.7

9.71

0.46

0.68

11.5

10.4

13.3

12.1

⅝

16

0.66

0.98

16.7

15.1

0.72

1.07

17.9

16.2

20.6

18.7

⅞

22

1.29

1.92

32.2

29.2

1.42

2.11

34.6

31.4

39.5

36.1

1⅛

29

2.13

3.17

52.6

47.7

2.34

3.48

56.5

51.3

65.0

59.0

1 3 / 8

35

3.18

4.73

77.7

70.5

3.50

5.21

83.5

75.7

96.0

87.1

1⅝

42

4.44

6.61

107

97.1

4.88

7.26

115

104

132

120

1⅞

48

5.91

8.8

141

128

6.5

9.67

152

138

174

158

2⅛

54

7.59

11.3

179

162

8.35

12.4

192

174

221

200

2 3 / 8

60

9.48

14.1

222

201

10.4

15.5

239

217

274

249

2⅞

67

11.6

17.3

268

243

12.8

19.0

288

261

331

300

3⅛

74

13.9

20.7

317

287

15.3

22.8

341

309

392

356

80

16.4

24.4

371

336

18.0

26.8

399

362

458

415

Open in new tab Share Figure 6.2.126 Values of Kbend vs. D/d ratios (D = sheave diameter, d = rope diameter), based on standard test data for 6 × 9 and 6 × 17 class ropes. (Compiled from "Wire Rope User's Manual," AISI, by permission.) Interactive Graph Values of Kbend vs. D/d ratios (D = sheave diameter, d = rope diameter), based on standard test data for 6 × 9 and 6 × 17 class ropes. (Compiled from "Wire Rope User's Manual," AISI, by permission.) Click on the graph to launch interactivity or enter values below. D/d ratio Kb Open in new tab Share Having made a tentative selection of a rope based on the demanded static load, one considers next the wear life of the rope. A loaded rope bent over a sheave stretches elastically and so rubs against the sheave, causing wear of both members. Drum or sheave size is of paramount importance at this point.

6.2.15.2. Sizing of Drums or Sheaves Diameters of drums or sheaves in wire rope applications are controlled by two main considerations: (1) the radial pressure between rope and groove and (2) degree of curvature imposed on the rope by the drum or sheave size.

Radial pressures can be calculated from p = 2T/(Dd), where p = unit radial pressure, lb/in2; T = rope load, lb; D = tread diameter of drum or sheave, in; d = nominal diameter of rope, in. Table 6.2.66 lists suggested allowable radial bearing pressures of ropes on various sheave materials.

Table 6.2.66 Suggested Allowable Radial Bearing Pressures of Ropes on Various Sheave Materials Material

Regular lay rope, lb/in2

Lang lay rope, lb/in2

Flattened strand lang lay, lb/in2

Remarks

6 × 7

6 × 19

6 × 37

8 × 19

6 × 7

6 × 19

6 × 37

Source: "Wire Rope User's Manual," AISI, reproduced by permission.

Wood

150

250

300

350

165

275

330

400

On end grain of beech, hickory, gum.

Cast iron

300

480

585

680

350

550

660

800

Based on minimum Brinell hardness of 125.

Carbon-steel casting

550

900

1,075

1,260

600

1,000

1,180

1,450

30-40 carbon. Based on minimum Brinell hardness of 160.

Chilled cast iron

650

1,100

1,325

1,550

715

1,210

1,450

1,780

Not advised unless surface is uniform in hardness.

Manganese steel

1,470

2,400

3,000

3,500

1,650

2,750

3,300

4,000

Grooves must be ground and sheaves balanced for high-speed service.

Open in new tab Share All wire ropes operating over drums or sheaves are subjected to cyclical stresses, causing shortened rope life because of fatigue. Fatigue resistance or relative service life is a function of the ratio D/d. Adverse effects also arise out of relative motion between strands during passage around the drum or sheave. Additional adverse effects can be traced to poor match between rope and groove size, and to lack of rope lubrication. Table 6.2.67 lists suggested and minimum sheave and drum ratios for various rope construction. Table 6.2.68 lists relative bending life factors; Figure 6.2.127 shows a plot of relative rope service life versus D/d. Table 6.2.69 lists minimum drum (sheave) groove dimensions. Periodic groove inspection is recommended, and worn or corrugated grooves should be re-machined or the drum replaced, depending on severity of damage.

Table 6.2.67 Sheave and Drum Ratios Construction* †

Suggested

Minimum

• WS—Warrington Seale; FWS—Filler Wire Seale; SFW—Seale Filler Wire; SWS—Seale Warrington Seale; S—Seale; FW—Filler Wire.

† D = tread diameter of sheave; d = nominal diameter of rope. To find any tread diameter from this table, the diameter for the rope construction to be used is multiplied by its nominal diameter d. For example, the minimum sheave tread diameter for a ½-in 6 × 21 FW rope would be ½ in (nominal diameter) × 30 (minimum ratio), or 15 in.

Note: These values are for reasonable service. Other values are permitted by various standards such as ANSI, API, PCSA, HMI, CMAA, etc. Similar values affect rope life.

Source: "Wire Rope User's Manual," AISI, reproduced by permission.

6 × 7

72

42

19 × 7 or 18 × 7 Rotation-resistant

51

34

6 × 19 S

51

34

6 × 25 B flattened strand

45

30

6 × 27 H flattened strand

45

30

6 × 30 G flattened strand

45

30

6 × 21 FW

45

30

6 × 26 WS

45

30

6 × 25 FW

39

26

6 × 31 WS

39

26

6 × 37 SFW

39

26

6 × 36 WS

35

23

6 × 43 FWS

35

23

6 × 41 WS

32

21

6 × 41 SFW

32

21

6 × 49 SWS

32

21

6 × 46 SFW

28

18

6 × 46 WS

28

18

8 × 19 S

41

27

8 × 25 FW

32

21

6 × 42 Tiller

21

14

Open in new tab Download data Share Table 6.2.68 Relative Bending Life Factors Rope construction

Factor

Rope construction

Factor

Source: "Wire Rope User's Manual," AISI, reproduced by permission.

6 × 7

0.61

6 × 36 WS

1.16

19 × 7 or 18 × 7

0.67

6 × 43 FWS

1.16

Rotation-resistant

0.81

6 × 41 WS

1.30

6 × 19 S

0.90

6 × 41 SFW

1.30

6 × 25 B flattened strand

0.90

6 × 49 SWS

1.30

6 × 27 H flattened strand

0.90

6 × 43 FW (2 op)

1.41

6 × 30 G flattened strand

0.89

6 × 46 SFW

1.41

6 × 21 FW

0.89

6 × 46 WS

1.41

6 × 26 WS

1.00

8 × 19 S

1.00

6 × 25 FW

1.00

8 × 25 FW

1.25

6 × 31 WS

1.00

6 × 42 Tiller

2.00

6 × 37 SFW

Open in new tab Download data Share Figure 6.2.127 Service life curves for various D/d ratios. Note that this curve takes into account only bending and tensile stresses. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) Interactive Graph Service life curves for various D/d ratios. Note that this curve takes into account only bending and tensile stresses. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) Click on the graph to launch interactivity or enter values below. D/d ratio Relative rope service life Open in new tab Share Table 6.2.69 Minimum Sheave- and Drum-Groove Dimensions* Nominal rope diameter

Groove radius

New

Worn

in

nm

in

mm

in

mm

• Values given are applicable to grooves in sheaves and drums; they are not generally suitable for pitch design since this may involve other factors. Further, the dimensions do not apply to traction-

type elevators; in this circumstance, drum- and sheave-groove tolerances should conform to the elevator manufacturer's specifications. Modern drum design embraces extensive considerations beyond the scope of this publication. It should also be noted that dram grooves are now produced with a number of oversize dimensions and pitches applicable to certain service requirements.

Source: "Wire Rope User's Manual," AISI, reproduced by permission.

¼

6.4

0.135

3.43

.129

3.28

5 / 16

8.0

0.167

4.24

.160

4.06

3 / 8

9.5

0.201

5.11

.190

4.83

7 / 16

11

0.234

5.94

.220

5.59

½

13

0.271

6.88

.256

6.50

9 / 16

14.5

0.303

7.70

.288

7.32

5 / 8

16

0.334

8.48

.320

8.13

3 / 4

19

0.401

10.19

.380

9.65

7 / 8

22

0.468

11.89

.440

11.18

1

26

0.543

13.79

.513

13.03

1⅛

29

0.605

15.37

.577

14.66

1¼

32

0.669

16.99

.639

16.23

1 3 / 8

35

0.736

18.69

.699

17.75

1½

38

0.803

20.40

.759

19.28

1⅝

42

0.876

22.25

.833

21.16

1¾

45

0.939

23.85

.897

22.78

1⅞

48

1.003

25.48

.959

24.36

2

52

1.085

27.56

1.025

26.04

2⅛

54

1.137

28.88

1.079

27.41

2¼

58

1.210

30.73

1.153

29.29

2 3 / 8

60

1.271

32.28

1.199

30.45

2½

64

1.338

33.99

1.279

32.49

2⅝

67

1.404

35.66

1.339

34.01

2¾

71

1.481

37.62

1.409

35.79

2⅞

74

1.544

39.22

1.473

37.41

3

77

1.607

40.82

1.538

39.07

3⅛

80

1.664

42.27

1.598

40.59

3¼

83

1.731

43.97

1.658

42.11

3 3 / 8

87

1.807

45.90

1.730

43.94

3½

90

1.869

47.47

1.794

45.57

3¾

96

1.997

50.72

1.918

48.72

4

103

2.139

54.33

2.050

52.07

4¼

109

2.264

57.51

2.178

55.32

4½

115

2.396

60.86

2.298

58.37

4¾

122

2.534

64.36

2.434

61.82

5

128

2.663

67.64

2.557

64.95

5¼

135

2.804

71.22

2.691

68.35

5½

141

2.929

74.40

2.817

71.55

5¾

148

3.074

78.08

2.947

74.85

6

154

3.198

81.23

3.075

78.11

Open in new tab Share Seizing and Cutting Wire Rope Before a wire rope is cut, seizings (bindings) must be applied on either side of the cut to prevent rope distortion and flattening or loosened strands. Normally, for preformed ropes, one seizing on each side of the cut is sufficient, but for ropes that are not preformed a minimum of two seizings on each side is recommended, and these should be spaced six rope diameters apart (see Fig. 6.2.128). Seizings should be made of soft or annealed wire or strand, and the width of the seizing should never be less than the diameter of the rope being seized. Table 6.2.70 lists suggested seizing wire diameters.

Figure 6.2.128 Seizings. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/b5689cae67236644/dabbb5c8ab916a67588e9e15144fa3607cde3a92171221b8c915b2690ccc4448/06x02_128.png Open in new tab Share Table 6.2.70 Seizing* Rope diameter

Suggested seizing wire diameter†

in

mm

in

mm

• Length of the seizing should not be less than the rope diameter.

† The diameter of seizing wire for elevator ropes is usually somewhat smaller than that shown in this table. Consult the wire rope manufacturer for specific size recommendations. Soft annealed seizing strand may also be used.

Source: "Wire Rope User's Manual," AISI, reproduced by permission.

⅛- 5 / 16

3.5-8.0

0.032

0.813

3 /

8

9 / 16

9.4-14.5

0.048

1.21

⅝- 15 / 16

16.0-24.0

0.063

1.60

1-1 5 / 16

26.0-33.0

0.080

2.03

1 3 / 8 -1 11 / 16

35.0-43.0

0.104

2.64

1¾ and larger

45.0 and larger

0.124

3.15

Open in new tab Share Wire Rope Fittings or Terminations End terminations allow forces to be transferred from rope to machine, or load to rope, etc. Figure 6.2.129 illustrates the most commonly used end fittings or terminations. Not all terminations will develop full strength. In fact, if all of the rope elements are not held securely, the individual strands will sustain unequal loads causing unequal wear among them, thus shortening the effective rope service life. Socketing allows an end fitting which reduces the chances of unequal strand loading.

Figure 6.2.129 End fittings, or terminations, showing the six most commonly used. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/93cbb5f808260c54/0b81d52ad105f0752c71c8e820d154a2b2caedd00b267e91c7d74013bc0b3bbc/06x02_129.png Open in new tab Share Wire rope manufacturers have developed a recommended procedure for socketing. A tight wire serving band is placed where the socket base will be, and the wires are unlaid, straightened, and "broomed" out. Fiber core is cut close to the serving band and removed, wires are cleaned with a solvent such as SC-methyl chloroform, and brushed to remove dirt and grease. If additional cleaning is done with muriatic acid this must be followed by a neutralizing rinse (if possible, ultrasonic cleaning is preferred). The wires are dipped in flux, the socket is positioned, zinc (spelter) is poured and allowed to set, the serving band is removed, and the rope lubricated.

A somewhat similar procedure is used in thermoset resin socketing.

Socketed terminations generally are able to develop 100 percent of nominal strength.

-uhogoyg

On 2014 Dec 15, Ivan Shatsky commented:

General comment: The phenomenon studied in this paper is very exciting. I read the article with a great interest. Unfortunately, I regret to say that the underlying mechanism remained uncovered. Although I agree that the authors identified some curious structures within the 5’UTRs of HOXA mRNAs which might be implicated in the regulation of these mRNAs by RPL38, I did not find sufficient evidence for existence of IRES-elements in these mRNAs. As in numerous other similar investigations, to identify IRES-elements the authors employed the method of DNA bicistronic constructs, the approach that had been repeatedly shown to be associated with almost unavoidable artifacts (see Jackson Cold Spring Harb Perspect Biol. 2013 Feb 1;5(2); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90.). And I suspect this paper is not free of those artifacts either (see below). Several crucial control experiments necessary to support or to exclude the IRES-mediated mechanism have been recently described (for references see Shatsky et al. Mol Cells. 2010 Oct; 30(4):285-93). One of them, for instance, is the ratio of translational activities for m7G capped versus uncapped (A-capped) monocistronic constructs. This value estimates contribution of the cap to the translational potential of a 5’UTR under selected conditions. If this contribution is very high (as is the case of cap-dependent mRNAs) one may exclude the presence of a true IRES. I think that this and other obligatory controls are feasible to perform with cells C3H10T1/2 used in this paper but they were not done.

Some specific points:

1. The authors regard the cap-independent and IRES-dependent modes of translation initiation as synonymous mechanisms and support this notion with reference 21. I should stress that not all specialists in eukaryotic translation would share this opinion since nobody has ever shown that a 5’end dependent translation initiation cannot be regulated by specific structures within the respective 5' UTR. The opposite has recently been demonstrated (Terenin et al. Nucleic Acids Res. 2013 Feb 1;41(3):1807-16.)
2. When listing examples of cellular IRESs identified to date (second paragraph), the authors mention c-myc, Apaf-1, XIAP. To the best of my knowledge, these cellular IRESs have been disproved (Andreev et al. Nucleic Acids Res. 2009 Oct;37(18):6135-47; Bert et al. RNA. 2006 Jun;12(6):1074-83; Baranick et al. Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4733-8; van Eden et al. RNA. 2004 Apr;10(4):720-30); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90. )
3. “Extended data Figure 1a” raises a great concern: the HCV IRES should not be used as a normalizing construct for testing bicistronic DNAs since the HCV IRES has been reported to harbor a cryptic promoter (Dumas, E. et al. 2003 Nucleic Acids Res. 31 (4): 1275-1281) and hence may produce capped monocistronic mRNAs . By the way, among viral IRESs characterized to date the HCV IRES is regarded as one of the weakest.
4. The control test with Rluc shRNA (Extended data Figure 1 b,c) strongly suggests that some significant amount of monocistronic (and therefore capped) Fluc mRNAs is present in transfected cells since the residual Fluc activity after RNA interference is too high. This may also be the case for the control bicistronic mRNA containing the HCV IRES (see point 3). Otherwise, the pictures 1b and 1c must be similar. At least, in the analogous test performed in our lab, the Rluc and Fluc activities fall down to the similar background levels (Fig. 2D in Dmitriev et al. Mol Cell Biol. 2007 Jul;27(13):4685-97).
5. The authors suggest that the IRES elements are mostly confined within ~300 nts proximal to the start codon. As a support to this conclusion, they note that some of HOXA mRNAs possess a 5’ UTR of that size. If so, why the activity of HOXA9 construct 944-1266 is much lower than that for the full length 5’UTR (Fig. 1d)?<br>
6. “Extended Figure 1d”. This control is useless. It only shows that the bicistronic mRNA of the expected size is present in transfected cells but unable to show the presence of monocistronic mRNAs starting within the intercistronic region. The corresponding bands won’t be seen.
7. On the base of pull-down experiments the authors claim that 80S ribosomes are specifically formed on their IRES-elements. The problem is that they use 10mM of magnesium in these expts, i.e. the concentration at which the assembly of translation initiation complexes in mammalian systems should not occur.
8. The mode of action of TIE element looks absolutely puzzling. It is even difficult to imagine any mechanism for its operation. My question: is it specific to these particular cells?

---

This comment, imported by Hypothesis from PubMed Commons, is licensed under CC BY.

On 2014 Dec 15, Ivan Shatsky commented:

General comment: The phenomenon studied in this paper is very exciting. I read the article with a great interest. Unfortunately, I regret to say that the underlying mechanism remained uncovered. Although I agree that the authors identified some curious structures within the 5’UTRs of HOXA mRNAs which might be implicated in the regulation of these mRNAs by RPL38, I did not find sufficient evidence for existence of IRES-elements in these mRNAs. As in numerous other similar investigations, to identify IRES-elements the authors employed the method of DNA bicistronic constructs, the approach that had been repeatedly shown to be associated with almost unavoidable artifacts (see Jackson Cold Spring Harb Perspect Biol. 2013 Feb 1;5(2); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90.). And I suspect this paper is not free of those artifacts either (see below). Several crucial control experiments necessary to support or to exclude the IRES-mediated mechanism have been recently described (for references see Shatsky et al. Mol Cells. 2010 Oct; 30(4):285-93). One of them, for instance, is the ratio of translational activities for m7G capped versus uncapped (A-capped) monocistronic constructs. This value estimates contribution of the cap to the translational potential of a 5’UTR under selected conditions. If this contribution is very high (as is the case of cap-dependent mRNAs) one may exclude the presence of a true IRES. I think that this and other obligatory controls are feasible to perform with cells C3H10T1/2 used in this paper but they were not done.

Some specific points:

1. The authors regard the cap-independent and IRES-dependent modes of translation initiation as synonymous mechanisms and support this notion with reference 21. I should stress that not all specialists in eukaryotic translation would share this opinion since nobody has ever shown that a 5’end dependent translation initiation cannot be regulated by specific structures within the respective 5' UTR. The opposite has recently been demonstrated (Terenin et al. Nucleic Acids Res. 2013 Feb 1;41(3):1807-16.)
2. When listing examples of cellular IRESs identified to date (second paragraph), the authors mention c-myc, Apaf-1, XIAP. To the best of my knowledge, these cellular IRESs have been disproved (Andreev et al. Nucleic Acids Res. 2009 Oct;37(18):6135-47; Bert et al. RNA. 2006 Jun;12(6):1074-83; Baranick et al. Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4733-8; van Eden et al. RNA. 2004 Apr;10(4):720-30); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90. )
3. “Extended data Figure 1a” raises a great concern: the HCV IRES should not be used as a normalizing construct for testing bicistronic DNAs since the HCV IRES has been reported to harbor a cryptic promoter (Dumas, E. et al. 2003 Nucleic Acids Res. 31 (4): 1275-1281) and hence may produce capped monocistronic mRNAs . By the way, among viral IRESs characterized to date the HCV IRES is regarded as one of the weakest.
4. The control test with Rluc shRNA (Extended data Figure 1 b,c) strongly suggests that some significant amount of monocistronic (and therefore capped) Fluc mRNAs is present in transfected cells since the residual Fluc activity after RNA interference is too high. This may also be the case for the control bicistronic mRNA containing the HCV IRES (see point 3). Otherwise, the pictures 1b and 1c must be similar. At least, in the analogous test performed in our lab, the Rluc and Fluc activities fall down to the similar background levels (Fig. 2D in Dmitriev et al. Mol Cell Biol. 2007 Jul;27(13):4685-97).
5. The authors suggest that the IRES elements are mostly confined within ~300 nts proximal to the start codon. As a support to this conclusion, they note that some of HOXA mRNAs possess a 5’ UTR of that size. If so, why the activity of HOXA9 construct 944-1266 is much lower than that for the full length 5’UTR (Fig. 1d)?<br>
6. “Extended Figure 1d”. This control is useless. It only shows that the bicistronic mRNA of the expected size is present in transfected cells but unable to show the presence of monocistronic mRNAs starting within the intercistronic region. The corresponding bands won’t be seen.
7. On the base of pull-down experiments the authors claim that 80S ribosomes are specifically formed on their IRES-elements. The problem is that they use 10mM of magnesium in these expts, i.e. the concentration at which the assembly of translation initiation complexes in mammalian systems should not occur.
8. The mode of action of TIE element looks absolutely puzzling. It is even difficult to imagine any mechanism for its operation. My question: is it specific to these particular cells?

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